Targeted mutant interferon-beta and uses thereof

ABSTRACT

The present invention relates, in part, to chimeric proteins comprising mutant interferon-β and their use as therapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/336,020, filed May 13, 2016, the entire contents ofall of which are herein incorporated by reference.

FIELD

The present invention relates, in part, to chimeric proteins comprisingmutant interferon-β and their use as therapeutic agents.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:ORN-019PC_Sequence_listing; date recorded: May 8, 2017; file size: 240KB).

BACKGROUND

Type I interferons (IFNs) form a family of multifunctional cytokinesthat play major roles in the immune response. The human type I IFNscomprises 13 distinct non-allelic alpha subtypes, one beta subtype, andone omega subtype. Differential activities of IFN subtypes have beenreported and used in the clinic for the treatment of variouspathologies, including viral hepatitis (IFN-α2) and multiple sclerosis(IFN-β).

IFNα and IFNβ are encoded by distinct genes and are independentlyregulated. As such, IFNβ is distinctly induced in response to externalsignals, such as pathogens and LPS of Gram-negative bacteria. IFNβ isalso selectively produced in response to signaling via the AP-1 pathwayby macrophage colony-stimulating factor and by the cytokine RANKL, andtherefore has a role in the development of myleloid cells andosteoclasts. These examples (and others) of selective production of IFNβindicates a function of IFNβ that is different relative to other type Iinterferons, including IFNα.

Further, numerous studies have shown that IFNβ can elicit signals incells that IFNα or other interferons cannot. For example, IFNβ, but notIFNα, induces the association of tyrosine-phosphorylated receptorcomponents IFNAR1 and IFNAR2, and has activity in cells lacking the IFNreceptor-associated, Janus kinase tyk2. Strikingly, IFNβ can elicitsignals and modulate gene expression in cells lacking IFNAR2. Theseeffects are mediated by binding of IFNβ to IFNAR1 in the absence ofIFNAR2. This unique property of IFNβ, as compared to IFNα or otherinterferons, is coupled with the ability of IFNβ to regulate theexpression of distinct genes—and underscores the unique biologicalactivity profile of IFNβ.

All type I IFNs are recognized by a single shared receptor composed oftwo transmembrane proteins, IFNAR1 and IFNAR2. Despite sequencedivergence, the type I IFNs interact with a common receptor. Numerousinstances of relative differences in activity have been noted. Some ofthese differences appear to be mediated by receptor binding selectivityat IFNAR1 and IFNAR2. For example, a prominent feature of IFN-β comparedto IFN-α2 is an ˜50-fold higher affinity towards IFNAR1.

A long held model for assembly of the interferon signal complex is thatinterferon initially binds IFNAR2 with the subsequent recruitment ofIFNAR1 to initiate signaling. An important difference between IFNβ andIFNα is that IFNα has high affinity for IFNAR2 and low affinity forIFNAR1. In contrast, IFNβ has high affinity for IFNAR2 and, compared toIFNα, a high affinity for IFNAR1. The differential binding affinitiesindicate that IFNβ has a distinct mechanism in receptor engagement ascompared to IFNα. Importantly, in alignment with biological data thatIFNβ can signal in cells that lack IFNAR2 but retain IFNAR1, the highaffinity of IFNβ for IFNAR1 may entail more than just a potentially moreefficient or functionally different signaling through the classicIFNAR1/2 complex.

Recent studies have further elucidated mechanisms underlying theactivity of IFNβ in cells deficient for IFNAR2. These studies show thatIFNβ can a) bind to IFNAR1 on cells in absence of IFNAR2, and b) thatIFNβ can bind to IFNAR1 extracellular domain in vitro with nanomolaraffinity. In parallel, the crystal structure of IFNβ bound to IFNAR1 ECDhas been solved. In contrast, IFNα cannot form a high affinity complexwith IFNAR1. Furthermore, analysis of the co-crystal structure of IFNβwith IFNAR1 revealed IFNβ-centric features (CD helix undergoes aconformational shift upon IFNAR1 binding and contributes a majorcomponent of the interaction with IFNAR1) not observed with otherinterferons in a IFNAR1/IFNAR2 bound state—providing an understandinghow IFNβ, but not other type of interferons, including IFNα, can bindIFNAR1 in the absence of IFNAR2. In other words, IFNβ but not IFNα cansignal through at least two different types of receptors: IFNAR1 (alone)and the IFNAR1/IFNAR2 complex.

The molecular difference between IFN-β and IFN-α is believed to beimportant therapeutically. For instance, a recent study providesevidence that IFNβ/IFNAR1 mediated signaling has in vivo biologicalsignificance. IFNβ is the only type I interferon induced by LPS, andcritical in mediating lethality in models of septic shock. IFNAR1deficient mice are protected from lethal effects of LPS challenge. Incontrast, IFNAR2 deficient mice demonstrate the same susceptibility toLPS-mediated toxicity as wild type mice. These findings suggest thatIFNβ signaling through IFNAR1 is likely involved in this pathogenicmechanism. Further administration of IFNβ to INFAR2-deficient miceexhibits toxicity, while IFNα does not.

Indeed, IFN-β has been prescribed for the treatment of different typesof cancers as well as other diseases including osteosarcoma, basal cellcarcinoma, cervical dysplasia, glioma, acute myeloid leukemia, multiplemyeloma, Hodgkin's disease, breast carcinoma, melanoma, viralinfections, and multiple sclerosis.

However, despite the benefits that it provides as compared to IFNα, thetherapeutic use of IFN-β is also associated with significant sideeffects such as pain, fever, chills, myalgias, arthralgias, and otherflu-like symptoms which narrow a patient's therapeutic window fortreatment and makes the patient more susceptible to other diseases.

Accordingly, there remains a need for safe and effective IFN-β-basedtherapeutics for treating cancer as well as other diseases and disorderswhile causing minimal toxicity and side effects.

SUMMARY

In some aspects, the present invention relates to chimeric proteinscomprising a modified interferon-beta (IFN-β) as a signaling agent. Inan embodiment, the IFN-β is a human IFN-β. In various embodiments, themodified IFN-β comprises one or more mutations that reduce itsbiological activity. In various embodiments, the modified IFN-βcomprises one or more mutations that reduce its affinity for atherapeutic receptor. In an embodiment, the therapeutic receptor is theinterferon-α/β receptor (IFNAR), which is composed of the IFNAR1 andIFNAR2 subunits. In an embodiment, the modified IFN-β comprises one ormore mutations that reduce its affinity for IFNAR1. In anotherembodiment, the modified IFN-β comprises one or more mutations thatreduce its affinity for IFNAR2. In an embodiment, the modified IFN-βcomprises one or more mutations that reduce its affinity for IFNAR2 andcomprises one or more mutations that reduce its affinity for IFNAR1.

In some embodiments, the chimeric protein comprises one or moreadditional signaling agents, e.g., without limitation, an interferon, aninterleukin, and a tumor necrosis factor, that may be modified. Invarious embodiments, the chimeric protein of the invention providesimproved safety compared to an unmodified, wildtype IFN-β.

In various embodiments, the chimeric protein comprises one or moretargeting moieties which have recognition domains (e.g. antigenrecognition domains, including without limitation various antibodyformats, inclusive of single-domain antibodies) which specifically bindto a target (e.g. antigen, receptor) of interest. In variousembodiments, the targeting moieties have recognition domains thatspecifically bind to a target (e.g. antigen, receptor) of interest,including those found on one or more immune cells, which can include,without limitation, T cells, cytotoxic T lymphocytes, T helper cells,natural killer (NK) cells, natural killer T (NKT) cells, anti-tumormacrophages (e.g. M1 macrophages), B cells, and dendritic cells. In someembodiments, the recognition domains specifically bind to a target (e.g.antigen, receptor) of interest and effectively recruit one of moreimmune cells. In some embodiments, the targets (e.g. antigens,receptors) of interest can be found on one or more tumor cells. In someembodiments, the present chimeric proteins may recruit an immune cell,e.g. an immune cell that can kill and/or suppress a tumor cell, to asite of action (such as, by way of non-limiting example, the tumormicroenvironment). In some embodiments, the recognition domainsspecifically bind to a target (e.g. antigen, receptor) of interest whichis part of a non-cellular structure.

In various embodiments, the present chimeric proteins find use in thetreatment of various diseases or disorders such as cancer, infections,immune disorders, autoimmune diseases, cardiovascular diseases, woundhealing, ischemia-related diseases, neurodegenerative diseases,metabolic diseases and many other diseases and disorders, and thepresent invention encompasses various methods of treatment.

In various embodiments, the present invention relates to the chimericproteins described herein for use as a medicament. In variousembodiments, the present invention relates to the chimeric proteins ofthe invention for use in treating one or more of cancer, infections,immune disorders, inflammatory diseases or conditions, and autoimmunediseases as described herein. Further, in various embodiments, any ofthe present agents may be for use in the treating, or the manufacture ofa medicament for treating, various diseases and disorders, including,but not limited to cancer, infections, immune disorders, inflammatorydiseases or conditions, and autoimmune diseases.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the anti-proliferative activity of various IFNβ-containingchimeras. Daudi and Wish cells were cultivated for three days with aserial dilution supernatants containing wild type IFNβ or variousIFNβ-containing chimeras (“AcTaferons”). Data are plotted as percentageof maximal proliferation, with error bars representing standarddeviation of triplicate measurements. Panels A-U show differentconstructs as described in the panel titles. For instance, Panel A is“IFNb,” i.e. wild type human IFN-β (SEQ ID NO: 1) without any fusion;Panel B is “VHH(CD20)-hIFNb(wt),” i.e. a fusion of a VHH against CD20with wild type human IFN-β (SEQ ID NO: 1); Panel C is“VHH(CD20)-hIFNb(R152A), i.e. a fusion of a VHH against CD20 with humanIFN-β having the R152A mutation (relative to SEQ ID NO: 1), and so on.

FIG. 2 shows the effects of various IFN-β-containing chimeras on STAT1phosphorylation. Mean fluorescence intensities of pSTAT1 in CD19positive and CD19 negative cells were plotted for wildtype IFN-β (i.e.SEQ ID NO: 1) or various IFNβ-containing chimeras (“AcTaferons,” PanelsA-I). Panels J-K show the mean fluorescence intensities of pSTAT1 forwildtype IFNβ or IFNβ-containing chimeras in CD19 positive cellsrelative to CD19 negative cells. Panels L-M show the targeting effectsof various IFNβ-containing chimeras relative to wildtype IFN-β. PanelsN-O show the targeting effects of various mutant IFNβ-containingchimeras relative to wildtype IFNβ-containing chimeras (a fusion of aVHH against CD20 with wild type human IFN-β (SEQ ID NO: 1)).

DETAILED DESCRIPTION

The present invention is based, in part, on the surprising discoverythat targeted chimeric proteins that include a modified IFN-β withreduced affinity for one or more receptors exhibit beneficialtherapeutic properties and reduced side effects. The present inventionprovides pharmaceutical compositions comprising the chimeric proteinsand their use in the treatment of various diseases. Administration ofthe chimeric proteins and pharmaceutical compositions of the inventionachieves significantly reduced side effects.

Modified Interferons

In one aspect, the present invention provides a chimeric protein thatincludes a signaling agent which is a modified version of an interferonwith reduced affinity for one or more receptors. Interferons (IFNs) area well-known family of cytokines secreted by a large variety ofeukaryotic cells. Interferons have a variety of biological activities,including anti-viral, immunomodulatory, immunoregulatory, andanti-proliferative properties, and have been utilized as therapeuticagents for treatment of various diseases. Interferons include a numberof related proteins, including interferon-alpha (IFN-α), interferon-beta(IFN-β), interferon-gamma IFN-γ) interferon-kappa (IFN-κ, also known asinterferon-epsilon or IFN-ε), interferon-tau (IFN-τ), andinterferon-omega (IFN-ω). These interferon proteins are produced in avariety of cell types: IFN-α (leukocytes), IFN-β (fibroblasts), IFN-γ(lymphocytes), IFN-ε or κ (keratinocytes), IFN-ω (leukocytes) and IFN-τ(trophoblasts). IFN-α, IFN-β, IFN-ε or κ, IFN-ω, and IFN-τ areclassified as type I interferons, while IFN-γ is classified as a type IIinterferon. Type-I interferons all appear to bind a common receptor,type I interferon-α/β receptor (IFNAR), composed of the IFNAR1 andIFNAR2 subunits. Upon binding of type I IFNs, IFNAR activates theJAK-STAT signaling pathway to elicit various biological effects.

In various embodiments, the chimeric protein of the invention comprisesa modified version of IFN-β as a signaling agent. In variousembodiments, the IFN-β encompasses functional derivatives, analogs,precursors, isoforms, splice variants, or fragments of IFN-β. In variousembodiments, the IFN-β encompasses IFN-β derived from any species. In anembodiment, the chimeric protein comprises a modified version of mouseIFN-β. In another embodiment, the chimeric protein comprises a modifiedversion of human IFN-β. Human IFN-β is a polypeptide with a molecularweight of about 22 kDa comprising 166 amino acid residues. The aminoacid sequence of human IFN-β is shown below:

(SEQ ID NO: 1) MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEI LRNFYFINRLTGYLRN.

In some embodiments, the human IFN-β is IFN-β-1a which is a glycosylatedform of human IFN-β. In some embodiments, the human IFN-β is IFN-β-1bwhich is a non-glycosylated form of human IFN-β that has a Met-1deletion and a Cys-17 to Ser mutation.

The sequences of IFN-β are known in the art. In various embodiments themodified IFN-β comprises an amino acid sequence that has at least about60%, or at least about 61%, or at least about 62%, or at least about63%, or at least about 64%, or at least about 65%, or at least about66%, or at least about 67%, or at least about 68%, or at least about69%, or at least about 70%, or at least about 71%, or at least about72%, or at least about 73%, or at least about 74%, or at least about75%, or at least about 76%, or at least about 77%, or at least about78%, or at least about 79%, or at least about 80%, or at least about81%, or at least about 82%, or at least about 83%, or at least about84%, or at least about 85%, or at least about 86%, or at least about87%, or at least about 88%, or at least about 89%, or at least about90%, or at least about 91%, or at least about 92%, or at least about93%, or at least about 94%, or at least about 95%, or at least about96%, or at least about 97%, or at least about 98%, or at least about 99%sequence identity with the known wild type amino acid sequences of IFN-β(e.g. about 60%, or about 61%, or about 62%, or about 63%, or about 64%,or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, orabout 70%, or about 71%, or about 72%, or about 73%, or about 74%, orabout 75%, or about 76%, or about 77%, or about 78%, or about 79%, orabout 80%, or about 81%, or about 82%, or about 83%, or about 84%, orabout 85%, or about 86%, or about 87%, or about 88%, or about 89%, orabout 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%, or about 96%, or about 97%, or about 98%, or about 99%sequence identity).

In some embodiments the modified IFN-β comprises an amino acid sequencethat has at least about 60%, or at least about 61%, or at least about62%, or at least about 63%, or at least about 64%, or at least about65%, or at least about 66%, or at least about 67%, or at least about68%, or at least about 69%, or at least about 70%, or at least about71%, or at least about 72%, or at least about 73%, or at least about74%, or at least about 75%, or at least about 76%, or at least about77%, or at least about 78%, or at least about 79%, or at least about80%, or at least about 81%, or at least about 82%, or at least about83%, or at least about 84%, or at least about 85%, or at least about86%, or at least about 87%, or at least about 88%, or at least about89%, or at least about 90%, or at least about 91%, or at least about92%, or at least about 93%, or at least about 94%, or at least about95%, or at least about 96%, or at least about 97%, or at least about98%, or at least about 99% sequence identity with human IFN-β having anamino acid sequence of SEQ ID NO:1 (e.g. about 60%, or about 61%, orabout 62%, or about 63%, or about 64%, or about 65%, or about 66%, orabout 67%, or about 68%, or about 69%, or about 70%, or about 71%, orabout 72%, or about 73%, or about 74%, or about 75%, or about 76%, orabout 77%, or about 78%, or about 79%, or about 80%, or about 81%, orabout 82%, or about 83%, or about 84%, or about 85%, or about 86%, orabout 87%, or about 88%, or about 89%, or about 90%, or about 91%, orabout 92%, or about 93%, or about 94%, or about 95%, or about 96%, orabout 97%, or about 98%, or about 99% sequence identity).

In various embodiments, the modified IFN-β comprises an amino acidsequence having one or more amino acid mutations. In some embodiments,the one or more amino acid mutations may be independently selected fromsubstitutions, insertions, deletions, and truncations.

In some embodiments, the amino acid mutations are amino acidsubstitutions, and may include conservative and/or non-conservativesubstitutions.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, IIe; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

In various embodiments, the substitutions may also include non-classicalamino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionineβ-alanine, GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA),D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β methyl amino acids, Cα-methyl amino acids, N α-methyl amino acids, and amino acid analogs ingeneral).

In various embodiments, the IFN-β is modified to have one or moremutations. In some embodiments, the mutations allow for the modifiedIFN-β to have one or more of attenuated activity such as one or more ofreduced binding affinity, reduced endogenous activity, and reducedspecific bioactivity relative to unmutated, e.g., the wild type form ofIFN-β. For instance, the one or more of attenuated activity such asreduced binding affinity, reduced endogenous activity, and reducedspecific bioactivity relative to unmutated, e.g. the wild type form ofIFN-β may be at a therapeutic receptor such as IFNAR. Consequentially,in various embodiments, the mutations allow for the modified solubleagent to have reduced systemic toxicity, reduced side effects, andreduced off-target effects relative to unmutated, e.g. the wild typeform of IFN-β.

In various embodiments, the IFN-β is modified to have a mutation thatreduces its binding affinity or activity at a therapeutic receptor suchas IFNAR. In some embodiments, the activity provided by the wild typeIFN-β is agonism at the therapeutic receptor (e.g. activation of acellular effect at a site of therapy). For example, the wild type IFN-βmay activate the therapeutic receptor. In such embodiments, the mutationresults in the modified IFN-β to have reduced activating activity at thetherapeutic receptor.

In some embodiments, the reduced affinity or activity at the therapeuticreceptor is restorable by attachment with a targeting moiety. In otherembodiments, the reduced affinity or activity at the therapeuticreceptor is not substantially restorable by attachment with thetargeting moiety. In various embodiments, the therapeutic chimericproteins of the present invention reduce off-target effects because theIFN-β has mutations that weaken binding affinity or activity at atherapeutic receptor. In various embodiments, this reduces side effectsobserved with, for example, the wild type IFN-β. In various embodiments,the modified IFN-β is substantially inactive en route to the site oftherapeutic activity and has its effect substantially on specificallytargeted cell types which greatly reduces undesired side effects.

In various embodiments, the modified IFN-β has one or more mutationsthat cause the IFN-β to have attenuated or reduced affinity, e.g.binding (e.g. K_(D)) and/or activation (measurable as, for example,K_(A) and/or EC₅₀) for one or more therapeutic receptors. In variousembodiments, the reduced affinity at the therapeutic receptor allows forattenuation of activity and/or signaling from the therapeutic receptor.

In various embodiments, the modified IFN-β has one or more mutationsthat reduce its binding to or its affinity for the IFNAR1 subunit ofIFNAR. In one embodiment, the modified IFN-β has reduced affinity and/oractivity at IFNAR1. In various embodiments, the modified IFN-β is humanIFN-β and has one or more mutations at positions F67, R71, L88, Y92,I95, N96, K123, and R124. In some embodiments, the one or more mutationsare substitutions selected from F67G, F67S, R71A, L88G, L88S, Y92G,Y92S, I95A, N96G, K123G, and R124G. In an embodiment, the modified IFN-βcomprises the F67G mutation. In an embodiment, the modified IFN-βcomprises the K123G mutation. In an embodiment, the modified IFN-βcomprises the F67G and R71A mutations. In an embodiment, the modifiedIFN-β comprises the L88G and Y92G mutations. In an embodiment, themodified IFN-β comprises the Y92G, I95A, and N96G mutations. In anembodiment, the modified IFN-β comprises the K123G and R124G mutations.In an embodiment, the modified IFN-β comprises the F67G, L88G, and Y92Gmutations. In an embodiment, the modified IFN-β comprises the F67S,L88S, and Y92S mutations.

In some embodiments, the modified IFN-β has one or more mutations thatreduce its binding to or its affinity for the IFNAR2 subunit of IFNAR.In one embodiment, the modified IFN-β has reduced affinity and/oractivity at IFNAR2. In various embodiments, the modified IFN-β is humanIFN-β and has one or more mutations at positions W22, R27, L32, R35,V148, L151, R152, and Y155. In some embodiments, the one or moremutations are substitutions selected from W22G, R27G, L32A, L32G, R35A,R35G, V148G, L151G, R152A, R152G, and Y155G. In an embodiment, themodified IFN-β comprises the W22G mutation. In an embodiment, themodified IFN-β comprises the L32A mutation. In an embodiment, themodified IFN-β comprises the L32G mutation. In an embodiment, themodified IFN-β comprises the R35A mutation. In an embodiment, themodified IFN-β comprises the R35G mutation. In an embodiment, themodified IFN-β comprises the V148G mutation. In an embodiment, themodified IFN-β comprises the R152A mutation. In an embodiment, themodified IFN-β comprises the R152G mutation. In an embodiment, themodified IFN-β comprises the Y155G mutation. In an embodiment, themodified IFN-β comprises the W22G and R27G mutations. In an embodiment,the modified IFN-β comprises the L32A and R35A mutation. In anembodiment, the modified IFN-β comprises the L151G and R152A mutations.In an embodiment, the modified IFN-β comprises the V148G and R152Amutations.

In some embodiments, the modified IFN-β has one or more of the followingmutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H.In some embodiments, the modified IFN-β has one or more of the followingmutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152Hin combination with C17S or C17A.

In some embodiments, the modified IFN-β has one or more of the followingmutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152Hin combination with any of the other IFN-β mutations described herein.

In some embodiments, the modified IFN-β has one or more mutations thatreduce its binding to or its affinity for both IFNAR1 and IFNAR2subunits.

In some embodiments, the modified IFN-β has one or more mutations thatreduce its binding to or its affinity for IFNAR1 and one or moremutations that substantially reduce or ablate binding to or its affinityfor IFNAR2. In some embodiments, chimeric proteins with such modifiedIFN-β can provide target-selective IFNAR1 activity (e.g. IFNAR1 activityis restorable via targeting through the targeting moiety).

In some embodiments, the modified IFN-β has one or more mutations thatreduce its binding to or its affinity for IFNAR1 and one or moremutations that reduce its binding to or its affinity for IFNAR2. In someembodiments, chimeric proteins with such modified IFN-β can providetarget-selective IFNAR1 and/or IFNAR2 activity (e.g. IFNAR1 and/IFNAR2activity is restorable via targeting through the targeting moiety).

The crystal structure of human IFN-β is known and is described inKarpusas et al., (1998) PNAS, 94(22): 11813-11818. Specifically, thestructure of human IFN-β has been shown to include five α-helices (i.e.,A, B, C, D, and E) and four loop regions that connect these helices(i.e., AB, BC, CD, and DE loops). In various embodiments, the modifiedIFN-β has one or more mutations in the A, B, C, D, E helices and/or theAB, BC, CD, and DE loops which reduce its binding affinity or activityat a therapeutic receptor such as IFNAR. Exemplary mutations aredescribed in WO2000/023114 and US20150011732, the entire contents ofwhich are hereby incorporated by reference. In an exemplary embodiment,the modified IFN-β is human IFN-β comprising alanine substitutions atamino acid positions 15, 16, 18, 19, 22, and/or 23. In an exemplaryembodiment, the modified IFN-β is human IFN-β comprising alaninesubstitutions at amino acid positions 28-30, 32, and 33. In an exemplaryembodiment, the modified IFN-β is human IFN-β comprising alaninesubstitutions at amino acid positions 36, 37, 39, and 42. In anexemplary embodiment, the modified IFN-β is human IFN-β comprisingalanine substitutions at amino acid positions 64 and 67 and a serinesubstitution at position 68. In an exemplary embodiment, the modifiedIFN-β is human IFN-β comprising alanine substitutions at amino acidpositions 71-73. In an exemplary embodiment, the modified IFN-β is humanIFN-β comprising alanine substitutions at amino acid positions 92, 96,99, and 100. In an exemplary embodiment, the modified IFN-β is humanIFN-β comprising alanine substitutions at amino acid positions 128, 130,131, and 134. In an exemplary embodiment, the modified IFN-β is humanIFN-β comprising alanine substitutions at amino acid positions 149, 153,156, and 159.

In various embodiments, the modified IFN-β has about 1%, or about 3%,about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%,about 20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100%of the affinity for the therapeutic receptor (e.g., IFNAR or any one ofits subunits IFNAR1 and/or IFNAR2) relative to the wild type IFN-β. Insome embodiments, the binding affinity is at least about 2-fold lower,about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-foldlower, about 7-fold lower, about 8-fold lower, about 9-fold lower, atleast about 10-fold lower, at least about 15-fold lower, at least about20-fold lower, at least about 25-fold lower, at least about 30-foldlower, at least about 35-fold lower, at least about 40-fold lower, atleast about 45-fold lower, at least about 50-fold lower, at least about100-fold lower, at least about 150-fold lower, or about 10-50-foldlower, about 50-100-fold lower, about 100-150-fold lower, about150-200-fold lower, or more than 200-fold lower relative to the wildtype IFN-β.

In various embodiments, the modified IFN-β comprises one or moremutations that reduce the endogenous activity of the IFN-β to about 75%,or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, orabout 25%, or about 20%, or about 10%, or about 5%, or about 3%, orabout 1%, e.g., relative to the wild type IFN-β.

In some embodiments, the modified IFN-β comprises one or more mutationsthat cause the modified IFN-β to have reduced affinity for a receptor.In some embodiments, the modified IFN-β's binding affinity for areceptor is lower than the binding affinity of the targeting moiety forits receptor. In some embodiments, this binding affinity differential isbetween the modified IFN-β/receptor and targeting moiety/receptor on thesame cell. In some embodiments, this binding affinity, differentialallows for the modified IFN-β to have localized, on-target effects andto minimize off-target effects that underlie side effects that areobserved with wild type IFN-β. In some embodiments, this bindingaffinity is at least about 2-fold, or at least about 5-fold, or at leastabout 10-fold, or at least about 15-fold lower, or at least about20-fold less, or at least about 25-fold, or at least about 30-fold less,or at least about 40-fold less, or at least about 50-fold lower, or atleast about 60-fold less, or at least about 70-fold less, or at leastabout 80-fold less, or at least about 90-fold less, or at least about100-fold, or at least about 150-fold less, or at least about 200-foldless, or at least about 250-fold less, or at least about 300-fold less,or at least about 350-fold less, or at least about 400-fold less, or atleast about 450-fold less, or at least about 500-fold less, or at leastabout 550-fold less, or at least about 600-fold less, or at least about650-fold less, or at least about 700-fold less, or at least about750-fold less, or at least about 800-fold less, or at least about850-fold less, or at least about 900-fold less, or at least about950-fold less, or at least about 1,000-fold less, or at least about1,100-fold less, or at least about 1,200-fold less, or at least about1,300-fold less, or at least about 1,400-fold less, or at least about1,500-fold less, or at least about 1,600-fold less, or at least about1,700-fold less, or at least about 1,800-fold less, or at least about1,900-fold less, or at least about 2,000-fold less.

In some embodiments, the modified IFN-β comprises one or more mutationsthat cause the modified IFN-β to have reduced endogenous activity orreduced specific bioactivity relative to unmutated, e.g., the wild typeform of IFN-β. In an exemplary embodiment, the reduced endogenousactivity or reduced specific bioactivity relate to anti-proliferativeactivity. In another exemplary embodiment, the reduced endogenousactivity or reduced specific bioactivity relate to STAT1phosphorylation. In some embodiments, endogenous activity or specificbioactivity is at least about 2-fold, or at least about 5-fold, or atleast about 10-fold, or at least about 15-fold lower, or at least about20-fold less, or at least about 25-fold, or at least about 30-fold less,or at least about 40-fold less, or at least about 50-fold lower, or atleast about 60-fold less, or at least about 70-fold less, or at leastabout 80-fold less, or at least about 90-fold less, or at least about100-fold, or at least about 150-fold less, or at least about 200-foldless, or at least about 250-fold less, or at least about 300-fold less,or at least about 350-fold less, or at least about 400-fold less, or atleast about 450-fold less, or at least about 500-fold less, or at leastabout 550-fold less, or at least about 600-fold less, or at least about650-fold less, or at least about 700-fold less, or at least about750-fold less, or at least about 800-fold less, or at least about850-fold less, or at least about 900-fold less, or at least about950-fold less, or at least about 1,000-fold less, or at least about1,100-fold less, or at least about 1,200-fold less, or at least about1,300-fold less, or at least about 1,400-fold less, or at least about1,500-fold less, or at least about 1,600-fold less, or at least about1,700-fold less, or at least about 1,800-fold less, or at least about1,900-fold less, or at least about 2,000-fold less. Receptor bindingactivity may be measured using methods known in the art. For example,affinity and/or binding activity may be assessed by Scatchard plotanalysis and computer-fitting of binding data (e.g. Scatchard, 1949) orby reflectometric interference spectroscopy under flow throughconditions, as described by Brecht et al. (1993), the entire contents ofall of which are hereby incorporated by reference.

In various embodiments, the attenuated activity at the therapeuticreceptor, the weakened affinity at the therapeutic receptor isrestorable by attachment with a targeting moiety, having high affinityfor an antigen at the site of therapeutic activity (e.g. an antibody orantibody format described herein). The targeting is realized by linkingthe modified IFN-β to a targeting moiety. In an embodiment, the modifiedIFN-β is linked to a targeting moiety through its amino-terminus. Inanother embodiment, the modified IFN-β is linked to a targeting moietythrough its carboxy-terminus. In this way, the present chimeric proteinsprovide, in some embodiments, localized, on-target, and controlledtherapeutic action at the therapeutic receptor.

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at W22, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at R27, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at W22, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V) and a mutation at R27, the mutation beingan aliphatic hydrophobic residue selected from glycine (G), alanine (A),leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at L32, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), isoleucine (I), methionine (M),and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at R35, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at L32, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), isoleucine (I), methionine (M),and valine (V) and a mutation at R35, the mutation being an aliphatichydrophobic residue selected from glycine (G), alanine (A), leucine (L),isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at F67, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at R71, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at F67, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V) and a mutation at R71, the mutation beingan aliphatic hydrophobic residue selected from glycine (G), alanine (A),leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at L88, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), isoleucine (I), methionine (M),and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at Y92, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at F67, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V) and a mutation at L88, the mutation beingan aliphatic hydrophobic residue selected from glycine (G), alanine (A),isoleucine (I), methionine (M), and valine (V) and a mutation at Y92,the mutation being an aliphatic hydrophobic residue selected fromglycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M),and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at L88, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), isoleucine (I), methionine (M),and valine (V) and a mutation at Y92, the mutation being an aliphatichydrophobic residue selected from glycine (G), alanine (A), leucine (L),isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at I95, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), methionine (M), andvaline (V) and a mutation at Y92, the mutation being an aliphatichydrophobic residue selected from glycine (G), alanine (A), leucine (L),isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at N96, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V) and a mutation at Y92, the mutation beingan aliphatic hydrophobic residue selected from glycine (G), alanine (A),leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at Y92, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V) and a mutation at I95, the mutation beingan aliphatic hydrophobic residue selected from glycine (G), alanine (A),leucine (L), methionine (M), and valine (V) and a mutation at N96, themutation being an aliphatic hydrophobic residue selected from glycine(G), alanine (A), leucine (L), isoleucine (I), methionine (M), andvaline (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at K123, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at R124, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at K123, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V) and a mutation at R124, the mutationbeing an aliphatic hydrophobic residue selected from glycine (G),alanine (A), leucine (L), isoleucine (I), methionine (M), and valine(V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at L151, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), isoleucine (I), methionine (M),and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at R152, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at L151, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), isoleucine (I), methionine (M),and valine (V) and a mutation at R152, the mutation being an aliphatichydrophobic residue selected from glycine (G), alanine (A), leucine (L),isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at V148, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I), andmethionine (M).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at V148, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V) and a mutation at R152, the mutationbeing an aliphatic hydrophobic residue selected from glycine (G),alanine (A), leucine (L), isoleucine (I), methionine (M), and valine(V).

In some embodiments, the mutant IFNβ comprises SEQ ID NO: 1 and amutation at Y155, the mutation being an aliphatic hydrophobic residueselected from glycine (G), alanine (A), leucine (L), isoleucine (I),methionine (M), and valine (V).

In some embodiments, the present invention relates to a chimeric proteincomprising: (a) a modified IFN-β, having the amino acid sequence of SEQID NO: 1 and a mutation at position W22, wherein the mutation is analiphatic hydrophobic residue; and (b) one or more targeting moieties,said targeting moieties comprising recognition domains whichspecifically bind to antigens or receptors of interest, the modifiedIFN-β and the one or more targeting moieties are optionally connectedwith one or more linkers. In various embodiments the mutation atposition W22 is aliphatic hydrophobic residue is selected from G, A, L,I, M, and V. In various embodiments the mutation at position W22 is G.

Therapeutic Agents Comprising the Present Modified Interferon-β

Targeting Moiety Cellular Recruitment

In various embodiments, the chimeric proteins of the present inventionadditionally comprise one or more targeting moieties having recognitiondomains which specifically bind to a target (e.g. antigen, receptor) ofinterest. In exemplary embodiments, the chimeric protein may comprisetwo, three, four, five, six, seven, eight, nine, ten or more targetingmoieties. In various embodiments, the target (e.g. antigen, receptor) ofinterest can be found on one or more immune cells, which can include,without limitation, T cells, cytotoxic T lymphocytes, T helper cells,natural killer (NK) cells, natural killer T (NKT) cells, anti-tumormacrophages (e.g. M1 macrophages), B cells, dendritic cells, or subsetsthereof. In some embodiments, the recognition domains specifically bindto a target (e.g. antigen, receptor) of interest and effectively,directly or indirectly, recruit one of more immune cells. In someembodiments, the target (e.g. antigen, receptor) of interest can befound on one or more tumor cells. In some embodiments, the chimericproteins of the invention comprise two or more targeting moieties. Insuch embodiments, the chimeric proteins can target two different cells(e.g. to make a synapse) or the same cell (e.g. to get a moreconcentrated signaling agent effect). In some embodiments, the presentchimeric proteins may directly or indirectly recruit an immune cell,e.g., in some embodiments, to a therapeutic site (e.g. a locus with oneor more disease cell or cell to be modulated for a therapeutic effect).In some embodiments, the present chimeric proteins may directly orindirectly recruit an immune cell, e.g. an immune cell that can killand/or suppress a tumor cell, to a site of action (such as, by way ofnon-limiting example, the tumor microenvironment).

In various embodiments, the targeting moieties can directly orindirectly recruit cells, such as disease cells and/or effector cells.In various embodiments, the signaling agent (e.g., modified IFN-β) canmodulate one or more cells that are targeted by the targeting moieties(e.g. recruited cells, such as disease cells and/or effector cells). Forinstance, the signaling agent (e.g., modified IFN-β) can modulate one orboth of the targeted cells (and the targeted cells can be effectorand/or disease cells), depending on whether the targeted cells express areceptor for the signaling agent.

In some embodiments, the present chimeric proteins are capable of, orfind use in methods involving, shifting the balance of immune cells infavor of immune attack of a tumor. For instance, the present chimericproteins can shift the ratio of immune cells at a site of clinicalimportance in favor of cells that can kill and/or suppress a tumor (e.g.T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK)cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g. M1macrophages), B cells, dendritic cells, or subsets thereof) and inopposition to cells that protect tumors (e.g. myeloid-derived suppressorcells (MDSCs), regulatory T cells (Tregs); tumor associated neutrophils(TANs), M2 macrophages, tumor associated macrophages (TAMs), or subsetsthereof). In some embodiments, the present chimeric protein is capableof increasing a ratio of effector T cells to regulatory T cells.

For example, in some embodiments, the recognition domains specificallybind to a target (e.g. antigen, receptor) associated with T cells. Insome embodiments, the recognition domains directly or indirectly recruitT cells. In an embodiment, the recognition domains specifically bind toeffector T cells. In some embodiments, the recognition domain directlyor indirectly recruits effector T cells, e.g., in some embodiments, to atherapeutic site (e.g. a locus with one or more disease cell or cell tobe modulated for a therapeutic effect). Illustrative effector T cellsinclude cytotoxic T cells (e.g. αβ TCR, CD3⁺, CD8⁺, CD45RO⁺); CD4⁺effector T cells (e.g. αβ TCR, CD3⁺, CD4⁺, CCR7⁺, CD62Lhi,IL-7R/CD127⁺); CD8⁺ effector T cells (e.g. αβ TCR, CD3⁺, CD8⁺, CCR7⁺,CD62Lhi, IL-7R/CD127 ⁺); effector memory T cells (e.g. CD62Llow, CD44⁺,TCR, CD3⁺, IL-7R/CD127⁺, IL-15R⁺, CCR7low); central memory T cells (e.g.CCR7⁺, CD62L⁺, CD27⁺; or CCR7hi, CD44⁺, CD62Lhi, TCR, CD3⁺,IL-7R/CD127⁺, IL-15R ⁺); CD62L⁺ effector T cells; CD8⁺ effector memory Tcells (TEM) including early effector memory T cells (CD27⁺ CD62L⁻) andlate effector memory T cells (CD27⁻ CD62L⁻) (TemE and TemL,respectively); CD127(⁺)CD25(low/−) effector T cells; CD127(⁻)CD25(⁻)effector T cells; CD8⁺ stem cell memory effector cells (TSCM) (e.g.CD44(low)CD62L(high)CD122(high)sca(⁺)); TH1 effector T-cells (e.g.CXCR3⁺, CXCR6⁺ and CCR5⁺; or αβ TCR, CD3⁺, CD4⁺, IL-12R⁺, IFNγR⁺,CXCR3⁺), TH2 effector T cells (e.g. CCR3⁺, CCR4⁺ and CCR8⁺; or αβ TCR,CD3⁺, CD4⁺, IL-4R⁺, IL-33R⁺, CCR4⁺, IL-17RB⁺, CRTH2⁺); TH9 effector Tcells (e.g. αβ TCR, CD3⁺, CD 4⁺); TH17 effector T cells (e.g. αβ TCR,CD3⁺, CD4⁺, IL-23R⁺, CCR6⁺, IL-1R⁺); CD4⁺CD45RO⁺CCR7 ⁺ effector T cells,ICOS⁺ effector T cells; CD4⁺CD45RO⁺CCR7(⁻) effector T cells; andeffector T cells secreting IL-2, IL-4 and/or IFN-γ.

Illustrative T cell antigens of interest include, for example (andinclusive of the extracellular domains, where applicable): CD8, CD3,SLAMF4, IL-2Rα, 4-1BB/TNFRSF9, IL-2 R β, ALCAM, B7-1, IL-4 R, B7-H3,BLAME/SLAMFS, CEACAM1, IL-6 R, CCR3, IL-7 Rα, CCR4, CXCRI/IL-S RA, CCR5,CCR6, IL-10R α, CCR 7, IL-I 0 R β, CCR5, IL-12 R β 1, CCR9, IL-12 R β 2,CD2, IL-13 R α 1, IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d,ILT5/CDS5a, lutegrin α 4/CD49d, CDS, Integrin α E/CD103, CD6, Integrin αM/CD 11 b, CDS, Integrin α X/CD11c, Integrin β 2/CDIS, KIR/CD15S,CD27/TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSFS, KIR2DL4/CD15Sd,CD31/PECAM-1, KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45,LAIR2, CDS3, Leukotriene B4-R1, CDS4/SLAMF5, NCAM-L1, CD94, NKG2A, CD97,NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6,Common γ Chain/IL-2 R γ, Osteopontin, CRACC/SLAMF7, PD-1, CRTAM, PSGL-1,CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, CXCR3, SIRP β 1,CXCR4, SLAM, CXCR6, TCCR/WSX-1, DNAM-1, Thymopoietin, EMMPRIN/CD147,TIM-1, EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, FcγRIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulysin, TNF RIII/TNFRSF1B, TRAILRI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102,TRAILR3/TNFRSF10C,IFN-γR1, TRAILR4/TNFRSF10D, IFN-γ R2, TSLP, IL-1 R1and TSLP R. In various embodiments, a targeting moiety of the chimericprotein binds one or more of these illustrative T cell antigens.

By way of non-limiting example, in various embodiments, the presentchimeric protein has a targeting moiety directed against a checkpointmarker expressed on a T cell, e.g. one or more of PD-1, CD28, CTLA4,ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR.

For example, in some embodiments, the recognition domains specificallybind to a target (e.g. antigen, receptor) associated with B cells. Insome embodiments, the recognition domains directly or indirectly recruitB cells, e.g., in some embodiments, to a therapeutic site (e.g. a locuswith one or more disease cell or cell to be modulated for a therapeuticeffect). Illustrative B cell antigens of interest include, for example,CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70,CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82,CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138 andCDw150. In various embodiments, a targeting moiety of the chimericprotein binds one or more of these illustrative B cell antigens.

By way of further example, in some embodiments, the recognition domainsspecifically bind to a target (e.g. antigen, receptor) associated withNatural Killer cells. In some embodiments, the recognition domainsdirectly or indirectly recruit Natural Killer cells, e.g., in someembodiments, to a therapeutic site (e.g. a locus with one or moredisease cell or cell to be modulated for a therapeutic effect).Illustrative Natural Killer cell antigens of interest include, forexample TIGIT, 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94,LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7, LMIR3/CD300LF, DNAM-1,LMIR5/CD300LB, Fc-epsilon RII, LMIR6/CD300LE, Fc-γ RI/CD64, MICA, Fc-γRIIB/CD32b, MICB, Fc-γ RIIC/CD32c, MULT-1, Fc-γ RIIA/CD32a,Nectin-2/CD112, Fc-γ RIII/CD16, NKG2A, FcRH1/IRTA5, NKG2C, FcRH2/IRTA4,NKG2D, FcRH4/IRTA1, NKp30, FcRH5/IRTA2, NKp44, Fc-Receptor-like3/CD16-2, NKp46/NCR1, NKp80/KLRF1, NTB-A/SLAMF6, Rae-1, Rae-1 α, Rae-1β, Rae-1 delta, H60, Rae-1 epsilon, ILT2/CD85j, Rae-1 γ, ILT3/CD85k,TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3, KIR/CD158, TREML1/TLT-1,KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4/CD158d and ULBP-3. In variousembodiments, a targeting moiety of the chimeric protein binds one ormore of these illustrative NK cell antigens.

Also, in some embodiments, the recognition domains specifically bind toa target (e.g. antigen, receptor) associated with macrophages/monocytes.In some embodiments, the recognition domains directly or indirectlyrecruit macrophages/monocytes, e.g., in some embodiments, to atherapeutic site (e.g. a locus with one or more disease cell or cell tobe modulated for a therapeutic effect). Illustrativemacrophages/monocyte antigens of interest include, for example SIRP1a,B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, Common β Chain, Integrin α4/CD49d, BLAME/SLAMF8, Integrin α X/CDIIc, CCL6/C10, Integrin β 2/CD18,CD155/PVR, Integrin β 3/CD61, CD31/PECAM-1, Latexin, CD36/SR-B3,Leukotriene B4 R1, CD40/TNFRSF5, LIMPIIISR-B2, CD43, LMIR1/CD300A, CD45,LMIR2/CD300c, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97,LMIR6/CD300LE, CD163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1,ECF-L, MD-2, EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB,Fc-γ RI/CD64, Osteopontin, Fc-γ RIIB/CD32b, PD-L2, Fc-γ RIIC/CD32c,Siglec-3/CD33, Fc-γ RIIA/CD32a, SIGNR1/CD209, Fc-γ RIII/CD16, SLAM,GM-CSF R α, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-γ RI, TLR4, IFN-gannnaR2, TREM-I, IL-I RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k,TREML1/TLT-1, 2B4/SLAMF 4, IL-10 R α, ALCAM, IL-10 R β,AminopeptidaseN/ANPEP, ILT2/CD85j, Common β Chain, ILT3/CD85k, ClqR1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, Integrin α 4/CD49d, CCR5,Integrin α M/CDII b, CCR8, Integrin α X/CDIIc, CD155/PVR, Integrin β2/CD18, CD14, Integrin β 3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45,Leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97,LMIR2/CD300c, LMIR3/CD300LF, Coagulation Factor III/Tissue Factor,LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1, CXCR6, M-CSFR, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR, Endoglin/CD105,NCAM-L1, Fc-γ RI/CD64, PSGL-1, Fc-γ RIIIICD16, RP105, G-CSF R,L-Selectin, GM-CSF R α, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54,TCCR/WSX-1, ICAM-2/CD102, TREM-I, IL-6 R, TREM-2, CXCRI/IL-8 RA, TREM-3and TREMLI/TLT-1. In various embodiments, a targeting moiety of thechimeric protein binds one or more of these illustrativemacrophage/monocyte antigens.

Also, in some embodiments, the recognition domains specifically bind toa target (e.g. antigen, receptor) associated with dendritic cells. Insome embodiments, the recognition domains directly or indirectly recruitdendritic cells, e.g., in some embodiments, to a therapeutic site (e.g.a locus with one or more disease cell or cell to be modulated for atherapeutic effect). Illustrative dendritic cell antigens of interestinclude, for example, CLEC9A, XCR1, RANK, CD36/SRB3, LOX-1/SR-E1, CD68,MARCO, CD163, SR-A1/MSR, CD5L, SREC-1, CL-PI/COLEC12, SREC-II,LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBBLigand/TNFSF9, IL-12/IL-23 p40, 4-Amino-1,8-naphthalimide, ILT2/CD85j,CCL21/6Ckine, ILT3/CD85k, 8-oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5,lutegrin α 4/CD49d, Aag, Integrin β 2/CD18, AMICA, Langerin, B7-2/CD86,Leukotriene B4 RI, B7-H3, LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, ClqR1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB CCR7, LMIR6/CD300LE,CD40/TNFRSF5, MAG/Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83,MDL-1/CLEC5A, CD84/SLAMF5, MMR, CD97, NCAMLI, CD2F-10/SLAMF9,Osteoactivin GPNMB, Chern 23, PD-L2, CLEC-1, RP105, CLEC-2, CLEC-8,Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33, DC-SIGN, Siglec-5,DC-SIGNR/CD299, Siglec-6, DCAR, Siglec-7, DCIR/CLEC4A, Siglec-9,DEC-205, Siglec-10, Dectin-1/CLEC7A, Siglec-F, Dectin-2/CLEC6A,SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147,TCCR/WSX-1, Fc-γ R1/CD64, TLR3, Fc-γ RIIB/CD32b, TREM-1, Fc-γRIIC/CD32c, TREM-2, Fc-γ RIIA/CD32a, TREM-3, Fc-γ RIII/CD16,TREML1/TLT-1, ICAM-2/CD102 and Vanilloid R1. In various embodiments, atargeting moiety of the chimeric protein binds one or more of theseillustrative DC antigens.

In some embodiments, the recognition domains specifically bind to atarget (e.g. antigen, receptor) on immune cells selected from, but notlimited to, megakaryocytes, thrombocytes, erythrocytes, mast cells,basophils, neutrophils, eosinophils, or subsets thereof. In someembodiments, the recognition domains directly or indirectly recruitmegakaryocytes, thrombocytes, erythrocytes, mast cells, basophils,neutrophils, eosinophils, or subsets thereof, e.g., in some embodiments,to a therapeutic site (e.g. a locus with one or more disease cell orcell to be modulated for a therapeutic effect).

In some embodiments, the recognition domains specifically bind to atarget (e.g. antigen, receptor) associated with megakaryocytes and/orthrombocytes. Illustrative megakaryocyte and/or thrombocyte antigens ofinterest include, for example, GP IIb/IIIa, GPIb, vWF, PF4, and TSP. Invarious embodiments, a targeting moiety of the chimeric protein bindsone or more of these illustrative megakaryocyte and/or thrombocyteantigens.

In some embodiments, the recognition domains specifically bind to atarget (e.g. antigen, receptor) associated with erythrocytes.Illustrative erythrocyte antigens of interest include, for example,CD34, CD36, CD38, CD41a (platelet glycoprotein IIb/IIIa), CD41b (GPIIb),CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit,HLA-DR, H2 (MHC-II), and Rhesus antigens. In In various embodiments, atargeting moiety of the chimeric protein binds one or more of theseillustrative erythrocyte antigens.

In some embodiments, the recognition domains specifically bind to atarget (e.g. antigen, receptor) associated with mast cells. Illustrativemast cells antigens of interest include, for example, SCFR/CD117,Fc_(ε)RI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMAI, FCERIA, FCER2,TPSABI. In various embodiments, a targeting moiety of the chimericprotein binds one or more of these mast cell antigens.

In some embodiments, the recognition domains specifically bind to atarget (e.g. antigen, receptor) associated with basophils. Illustrativebasophils antigens of interest include, for example, Fc_(ε)RI, CD203c,CD123, CD13, CD107a, CD107b, and CD164. In various embodiments, atargeting moiety of the chimeric protein binds one or more of thesebasophil antigens.

In some embodiments, the recognition domains specifically bind to atarget (e.g. antigen, receptor) associated with neutrophils.Illustrative neutrophils antigens of interest include, for example, 7D5,CD10/CALLA, CD13, CD16 (FcRIII), CD18 proteins (LFA-1, CR3, and p150,95), CD45, CD67, and CD177. In various embodiments, a targeting moietyof the chimeric protein binds one or more of these neutrophil antigens.

In some embodiments, the recognition domains specifically bind to atarget (e.g. antigen, receptor) associated with eosinophils.Illustrative eosinophils antigens of interest include, for example,CD35, CD44 and CD69. In various embodiments, a targeting moiety of thechimeric protein binds one or more of these eosinophil antigens.

In various embodiments, the recognition domain may bind to anyappropriate target, antigen, receptor, or cell surface markers known bythe skilled artisan. In some embodiments, the antigen or cell surfacemarker is a tissue-specific marker. Illustrative tissue-specific markersinclude, but are not limited to, endothelial cell surface markers suchas ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAMI, PROCR, SELF,SELP, TEK, THBD, VCAMI, VWF; smooth muscle cell surface markers such asACTA2, MYHIO, MYHI 1, MYH9, MYOCD; fibroblast (stromal) cell surfacemarkers such as ALCAM, CD34, COLIAI, COL1A2, COL3A1, FAP, PH-4;epithelial cell surface markers such as CDID, K6IRS2, KRTIO, KRT13,KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUCI, TACSTDI; neovasculaturemarkers such as CD13, TFNA, Alpha-v beta-3 (α_(v)β₃), E-selectin; andadipocyte surface markers such as ADIPOQ, FABP4, and RETN. In variousembodiments, a targeting moiety of the chimeric protein binds one ormore of these antigens. In various embodiments, a targeting moiety ofthe chimeric protein binds one or more of cells having these antigens.

In some embodiments, the recognition domains specifically bind to atarget (e.g. antigen, receptor) associated with tumor cells. In someembodiments, the recognition domains directly or indirectly recruittumor cells. For instance, in some embodiments, the direct or indirectrecruitment of the tumor cell is to one or more effector cell (e.g. animmune cell as described herein) that can kill and/or suppress the tumorcell.

Tumor cells, or cancer cells refer to an uncontrolled growth of cells ortissues and/or an abnormal increased in cell survival and/or inhibitionof apoptosis which interferes with the normal functioning of bodilyorgans and systems. For example, tumor cells include benign andmalignant cancers, polyps, hyperplasia, as well as dormant tumors ormicrometastases. Illustrative tumor cells include, but are not limitedto cells of: basal cell carcinoma, biliary tract cancer; bladder cancer;bone cancer; brain and central nervous system cancer; breast cancer;cancer of the peritoneum; cervical cancer; choriocarcinoma; colon andrectum cancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm;kidney or renal cancer; larynx cancer; leukemia; liver cancer; lungcancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue,mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as wellas B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (e.g. that associated with brain tumors), andMeigs' syndrome.

Tumor cells, or cancer cells also include, but are not limited to,carcinomas, e.g. various subtypes, including, for example,adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, andtransitional cell carcinoma), sarcomas (including, for example, bone andsoft tissue), leukemias (including, for example, acute myeloid, acutelymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell),lymphomas and myelomas (including, for example, Hodgkin and non-Hodgkinlymphomas, light chain, non-secretory, MGUS, and plasmacytomas), andcentral nervous system cancers (including, for example, brain (e.g.gliomas (e.g. astrocytoma, oligodendroglioma, and ependymoma),meningioma, pituitary adenoma, and neuromas, and spinal cord tumors(e.g. meningiomas and neurofibroma).

Illustrative tumor antigens include, but are not limited to,MART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosinedeaminase-binding protein (ADAbp), cyclophilin b, Colorectal associatedantigen (CRC)-0017-1A/GA733, Carcinoembryonic Antigen (CEA) and itsimmunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate SpecificAntigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3,prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zetachain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family oftumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4,tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein,E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117,PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC),fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides,viral products such as human papilloma virus proteins, Smad family oftumor antigens, Imp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brainglycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5,SCP-1 CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD56, CD70,CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, and PMSA. Invarious embodiments, a targeting moiety of the chimeric protein bindsone or more of these tumor antigens.

In various embodiments, the chimeric proteins of the invention havetargeting moieties having recognition domains which specifically bind toa target (e.g. antigen, receptor) which is part of a non-cellularstructure. In some embodiments, the antigen or receptor is not anintegral component of an intact cell or cellular structure. In someembodiments, the antigen or receptor is an extracellular antigen orreceptor. In some embodiments, the target is a non-proteinaceous,non-cellular marker, including, without limitation, nucleic acids,inclusive of DNA or RNA, such as, for example, DNA released fromnecrotic tumor cells or extracellular deposits such as cholesterol.

In some embodiments, the target (e.g. antigen, receptor) of interest ispart of the non-cellular component of the stroma or the extracellularmatrix (ECM) or the markers associated therewith. As used herein, stromarefers to the connective and supportive framework of a tissue or organ.Stroma may include a compilation of cells such asfibroblasts/myofibroblasts, glial, epithelia, fat, immune, vascular,smooth muscle, and immune cells along with the extracellular matrix(ECM) and extracellular molecules. In various embodiments, the target(e.g. antigen, receptor) of interest is part of the non-cellularcomponent of the stroma such as the extracellular matrix andextracellular molecules. As used herein, the ECM refers to thenon-cellular components present within all tissues and organs. The ECMis composed of a large collection of biochemically distinct componentsincluding, without limitation, proteins, glycoproteins, proteoglycans,and polysaccharides. These components of the ECM are usually produced byadjacent cells and secreted into the ECM via exocytosis. Once secreted,the ECM components often aggregate to form a complex network ofmacromolecules. In various embodiments, the chimeric protein of theinvention comprises a targeting moiety that recognizes a target (e.g.,an antigen or receptor or non-proteinaceous molecule) located on anycomponent of the ECM. Illustrative components of the ECM include,without limitation, the proteoglycans, the non-proteoglycanpolysaccharides, fibers, and other ECM proteins or ECM non-proteins,e.g. polysaccharides and/or lipids, or ECM associated molecules (e.g.proteins or non-proteins, e.g. polysaccharides, nucleic acids and/orlipids).

In some embodiments, the targeting moiety recognizes a target (e.g.antigen, receptor) on ECM proteoglycans. Proteoglycans are glycosylatedproteins. The basic proteoglycan unit includes a core protein with oneor more covalently attached glycosaminoglycan (GAG) chains.Proteoglycans have a net negative charge that attracts positivelycharged sodium ions (Na+), which attracts water molecules via osmosis,keeping the ECM and resident cells hydrated. Proteoglycans may also helpto trap and store growth factors within the ECM. Illustrativeproteoglycans that may be targeted by the chimeric proteins of theinvention include, but are not limited to, heparan sulfate, chondroitinsulfate, and keratan sulfate. In an embodiment, the targeting moietyrecognizes a target (e.g. antigen, receptor) on non-proteoglycanpolysaccharides such as hyaluronic acid.

In some embodiments, the targeting moiety recognizes a target (e.g.antigen, receptor) on ECM fibers. ECM fibers include collagen fibers andelastin fibers. In some embodiments, the targeting moiety recognizes oneor more epitopes on collagens or collagen fibers. Collagens are the mostabundant proteins in the ECM. Collagens are present in the ECM asfibrillar proteins and provide structural support to resident cells. Inone or more embodiments, the targeting moiety recognizes and binds tovarious types of collagens present within the ECM including, withoutlimitation, fibrillar collagens (types I, II, III, V, XI), facitcollagens (types IX, XII, XIV), short chain collagens (types VIII, X),basement membrane collagens (type IV), and/or collagen types VI, VII, orXIII. Elastin fibers provide elasticity to tissues, allowing them tostretch when needed and then return to their original state. In someembodiments, the target moiety recognizes one or more epitopes onelastins or elastin fibers.

In some embodiments, the targeting moiety recognizes one or more ECMproteins including, but not limited to, a tenascin, a fibronectin, afibrin, a laminin, or a nidogen/entactin.

In an embodiment, the targeting moiety recognizes and binds to tenascin.The tenascin (TN) family of glycoproteins includes at least fourmembers, tenascin-C, tenascin-R, tenascin-X, and tenascin W. The primarystructures of tenascin proteins include several common motifs ordered inthe same consecutive sequence: amino-terminal heptad repeats, epidermalgrowth factor (EGF)-like repeats, fibronectin type III domain repeats,and a carboxyl-terminal fibrinogen-like globular domain. Each proteinmember is associated with typical variations in the number and nature ofEGF-like and fibronectin type III repeats. Isoform variants also existparticularly with respect to tenascin-C. Over 27 splice variants and/orisoforms of tenascin-C are known. In a particular embodiment, thetargeting moiety recognizes and binds to tenascin-CA1. Similarly,tenascin-R also has various splice variants and isoforms. Tenascin-Rusually exists as dimers or trimers. Tenascin-X is the largest member ofthe tenascin family and is known to exist as trimers. Tenascin-W existsas trimers. In some embodiments, the targeting moiety recognizes one ormore epitopes on a tenascin protein. In some embodiments, the targetingmoiety recognizes the monomeric and/or the dimeric and/or the trimericand/or the hexameric forms of a tenascin protein.

In an embodiment, the targeting moieties recognize and bind tofibronectin. Fibronectins are glycoproteins that connect cells withcollagen fibers in the ECM, allowing cells to move through the ECM. Uponbinding to integrins, fibronectins unfolds to form functional dimers. Insome embodiments, the targeting moiety recognizes the monomeric and/orthe dimeric forms of fibronectin. In some embodiments, the targetingmoiety recognizes one or more epitopes on fibronectin. In illustrativeembodiments, the targeting moiety recognizes fibronectin extracellulardomain A (EDA) or fibronectin extracellular domain B (EDB). Elevatedlevels of EDA are associated with various diseases and disordersincluding psoriasis, rheumatoid arthritis, diabetes, and cancer. In someembodiments, the targeting moiety recognizes fibronectin that containsthe EDA isoform and may be utilized to target the chimeric protein todiseased cells including cancer cells. In some embodiments, thetargeting moiety recognizes fibronectin that contains the EDB isoform.In various embodiments, such targeting moieties may be utilized totarget the chimeric protein to tumor cells including the tumorneovasculature.

In an embodiment, the targeting moiety recognizes and binds to fibrin.Fibrin is another protein substance often found in the matrix network ofthe ECM. Fibrin is formed by the action of the protease thrombin onfibrinogen which causes the fibrin to polymerize. In some embodiments,the targeting moiety recognizes one or more epitopes on fibrin. In someembodiments, the targeting moiety recognizes the monomeric as well asthe polymerized forms of fibrin.

In an embodiment, the targeting moiety recognizes and binds to laminin.Laminin is a major component of the basal lamina, which is a proteinnetwork foundation for cells and organs. Laminins are heterotrimericproteins that contain an α-chain, β-chain, and a γ-chain. In someembodiments, the targeting moiety recognizes one or more epitopes onlaminin. In some embodiments, the targeting moiety recognizes themonomeric, the dimeric as well as the trimeric forms of laminin.

In an embodiment, the targeting moiety recognizes and binds to a nidogenor entactin. Nidogens/entactins are a family of highly conserved,sulfated glycoproteins. They make up the major structural component ofthe basement membranes and function to link laminin and collagen IVnetworks in basement membranes. Members of this family include nidogen-1and nidogen-2. In various embodiments, the targeting moiety recognizesan epitope on nidogen-1 and/or nidogen-2.

In various embodiments, the targeting moiety comprises an antigenrecognition domain that recognizes an epitope present on any of thetargets (e.g., ECM proteins) described herein. In an embodiment, theantigen-recognition domain recognizes one or more linear epitopespresent on the protein. As used herein, a linear epitope refers to anycontinuous sequence of amino acids present on the protein. In anotherembodiment, the antigen-recognition domain recognizes one or moreconformational epitopes present on the protein. As used herein, aconformation epitope refers to one or more sections of amino acids(which may be discontinuous) which form a three-dimensional surface withfeatures and/or shapes and/or tertiary structures capable of beingrecognized by an antigen recognition domain.

In various embodiments, the targeting moiety may bind to the full-lengthand/or mature forms and/or isoforms and/or splice variants and/orfragments and/or any other naturally occurring or synthetic analogs,variants, or mutants of any of the targets (e.g., ECM proteins)described herein. In various embodiments, the targeting moiety may bindto any forms of the proteins described herein, including monomeric,dimeric, trimeric, tetrameric, heterodimeric, multimeric and associatedforms. In various embodiments, the targeting moiety may bind to anypost-translationally modified forms of the proteins described herein,such as glycosylated and/or phosphorylated forms.

In various embodiments, the targeting moiety comprises an antigenrecognition domain that recognizes extracellular molecules such as DNA.In some embodiments, the targeting moiety comprises an antigenrecognition domain that recognizes DNA. In an embodiment, the DNA isshed into the extracellular space from necrotic or apoptotic tumor cellsor other diseased cells.

In various embodiments, the targeting moiety comprises an antigenrecognition domain that recognizes one or more non-cellular structuresassociated with atherosclerotic plaques. Two types of atheroscleroticplaques are known. The fibro-lipid (fibro-fatty) plaque is characterizedby an accumulation of lipid-laden cells underneath the intima of thearteries. Beneath the endothelium there is a fibrous cap covering theatheromatous core of the plaque. The core includes lipid-laden cells(macrophages and smooth muscle cells) with elevated tissue cholesteroland cholesterol ester content, fibrin, proteoglycans, collagen, elastin,and cellular debris. In advanced plaques, the central core of the plaqueusually contains extracellular cholesterol deposits (released from deadcells), which form areas of cholesterol crystals with empty, needle-likeclefts. At the periphery of the plaque are younger foamy cells andcapillaries. A fibrous plaque is also localized under the intima, withinthe wall of the artery resulting in thickening and expansion of the walland, sometimes, spotty localized narrowing of the lumen with someatrophy of the muscular layer. The fibrous plaque contains collagenfibers (eosinophilic), precipitates of calcium (hematoxylinophilic) andlipid-laden cells. In some embodiments, the targeting moiety recognizesand binds to one or more of the non-cellular components of these plaquessuch as the fibrin, proteoglycans, collagen, elastin, cellular debris,and calcium or other mineral deposits or precipitates. In someembodiments, the cellular debris is a nucleic acid, e.g. DNA or RNA,released from dead cells.

In various embodiments, the targeting moiety comprises an antigenrecognition domain that recognizes one or more non-cellular structuresfound in the brain plaques associated with neurodegenerative diseases.In some embodiments, the targeting moiety recognizes and binds to one ormore non-cellular structures located in the amyloid plaques found in thebrains of patients with Alzheimer's disease. For example, the targetingmoiety may recognize and bind to the peptide amyloid beta, which is amajor component of the amyloid plaques. In some embodiments, thetargeting moiety recognizes and binds to one or more non-cellularstructures located in the brains plaques found in patients withHuntington's disease. In various embodiments, the targeting moietyrecognizes and binds to one or more non-cellular structures found inplaques associated with other neurodegenerative or musculoskeletaldiseases such as Lewy body dementia and inclusion body myositis.

In some embodiments, the present chimeric protein comprises two or moretargeting moieties. In some embodiments, the present chimeric proteinhas (i) one or more targeting moieties directed against an immune cellselected from a T cell, a B cell, a dendritic cell, a macrophage, a NKcell, or subsets thereof and (ii) one or more targeting moietiesdirected against a tumor cell, along with any of the modified (e.g.mutant) signaling agents described herein (e.g., modified IFN-β). In oneembodiment, the present chimeric protein has (i) a targeting moietydirected against a T cell (including, without limitation an effector Tcell) and (ii) a targeting moiety is directed against a tumor cell,along with any of the modified (e.g. mutant) signaling agents describedherein (e.g., modified IFN-β). In one embodiment, the present chimericprotein has (i) a targeting moiety directed against a B cell and (ii) atargeting moiety is directed against a tumor cell, along with any of themodified (e.g. mutant) signaling agents described herein (e.g., modifiedIFN-β). In one embodiment, the present chimeric protein has (i) atargeting moiety directed against a dendritic cell and (ii) a targetingmoiety is directed against a tumor cell, along with any of the modified(e.g. mutant) signaling agents described herein (e.g., modified IFN-β).In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a macrophage and (ii) a targeting moiety isdirected against a tumor cell, along with any of the modified (e.g.mutant) signaling agents described herein (e.g., modified IFN-β). In oneembodiment, the present chimeric protein has (i) a targeting moietydirected against a NK cell and (ii) a targeting moiety is directedagainst a tumor cell, along with any of the modified (e.g. mutant)signaling agents described herein (e.g., modified IFN-β).

By way of non-limiting example, in various embodiments, the presentchimeric protein has (i) a targeting moiety directed against a T cell,for example, mediated by targeting to CD8, SLAMF4, IL-2 R α,4-1BB/TNFRSF9, IL-2 R β, ALCAM, B7-1, IL-4 R, B7-H3, BLAME/SLAMFS,CEACAM1, IL-6 R, CCR3, IL-7 Rα, CCR4, CXCRI/IL-S RA, CCR5, CCR6, IL-10Rα, CCR 7, IL-I 0 R β, CCR5, IL-12 R β 1, CCR9, IL-12 R β 2, CD2, IL-13 Rα 1, IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5a,lutegrin α 4/CD49d, CDS, Integrin α E/CD103, CD6, Integrin α M/CD 11 b,CDS, Integrin α X/CD11c, Integrin β 2/CDIS, KIR/CD15S, CD27/TNFRSF7,KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSFS, KIR2DL4/CD15Sd, CD31/PECAM-1,KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3,Leukotriene B4-R1, CDS4/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C,CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common γChain/IL-2 R γ, Osteopontin, CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4,RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, CXCR3, SIRP β 1, CXCR4,SLAM, CXCR6, TCCR/WSX-1, DNAM-1, Thymopoietin, EMMPRIN/CD147, TIM-1,EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, FcγRIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulysin, TNF RIII/TNFRSF1B, TRAILRI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102,TRAILR3/TNFRSF10C, IFN-γR1, TRAILR4/TNFRSF10D, IFN-γ R2, TSLP, IL-1 R1,or TSLP R; and (ii) a targeting moiety is directed against a tumor cell,along with any of the modified (e.g. mutant) signaling agents describedherein (e.g., modified IFN-β).

By way of non-limiting example, in various embodiments, the presentchimeric protein has a targeting moiety directed against (i) acheckpoint marker expressed on a T cell, e.g. one or more of PD-1, CD28,CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, Cd27, CD40L, TIM3, and A2aRand (ii) a targeting moiety is directed against a tumor cell, along withany of the modified (e.g. mutant) signaling agents described herein(e.g., modified IFN-β).

In various embodiments, the present chimeric protein has one or moretargeting moieties directed against PD-1. In some embodiments, thechimeric protein has one or more targeting moieties which selectivelybind a PD-1 polypeptide. In some embodiments, the chimeric proteincomprises one or more antibodies, antibody derivatives or formats,peptides or polypeptides, or fusion proteins that selectively bind aPD-1 polypeptide.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibodypembrolizumab (aka MK-3475, KEYTRUDA), or fragments thereof.Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed inHamid, et al. (2013) New England Journal of Medicine 369 (2): 134-44,U.S. Pat. No. 8,354,509, and WO 2009/114335, the entire disclosures ofwhich are hereby incorporated by reference. In illustrative embodiments,pembrolizumab or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising the aminoacid sequence of:

(SEQ ID NO: 2) QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 3) EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody,nivolumab (aka BMS-936558, MDX-1106, ONO-4538, OPDIVO), or fragmentsthereof. Nivolumab (clone 5C4) and other human monoclonal antibodiesthat specifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449and WO 2006/121168, the entire disclosures of which are herebyincorporated by reference. In illustrative embodiments, nivolumab or anantigen-binding fragment thereof comprises a heavy chain comprising theamino acid sequence of:

(SEQ ID NO: 4) QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAVIWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVTVSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVLQSSGLYSLSS VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRWQEGNVFSCSV MHEALHNHYT QKSLSLSLGK;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 5) EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibodypidilizumab (aka CT-011, hBAT or hBAT-1), or fragments thereof.Pidilizumab and other humanized anti-PD-I monoclonal antibodies aredisclosed in US 2008/0025980 and WO 2009/101611, the entire disclosuresof which are hereby incorporated by reference. In illustrativeembodiments, the anti-PD-1 antibody or an antigen-binding fragmentthereof for use in the methods provided herein comprises a light chainvariable regions comprising an amino acid sequence selected from SEQ IDNOS: 15-18 of US 2008/0025980:

SEQ ID No: 15 of US 2008/0025980: (SEQ ID NO: 6)EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQRSSFPLTFGGG TKLEIK; SEQ ID No: 16of US 2008/0025980: (SEQ ID NO: 7)EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTDYTLTINSLQPEDFATYYCQQRSSFPLTFGGG TKLEIK; SEQ ID No: 17of US 2008/0025980: (SEQ ID NO: 8)EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTDYCLTINSLQPEDFATYYCQQRSSFPLTFGGG TKLEIK; SEQ ID No: 18of US 2008/0025980: (SEQ ID NO: 9)EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTFGGG TKLEIK;

and/or a heavy chain comprising an amino acid sequence selected from SEQID NOS: 20-24 of US 2008/0025980:

SEQ ID No: 20 of US 2008/0025980: (SEQ ID NO: 10)QVQLVQSGSELKKPGASVKISCKASGYSFSNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVSTAYLQITSLTAEDTGMYFCAKVG YDALDYWGQGTLVTVSS;SEQ ID No: 21 of US 2008/0025980: (SEQ ID NO: 11)QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVSTAYLQITSLTAEDTGMYFCAKVG YDALDYWGQGTLVTVSS;SEQ ID No: 22 of US 2008/0025980: (SEQ ID NO: 12)QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVG YDALDYWGQGTLVTVSS;SEQ ID No: 23 of US 2008/0025980: (SEQ ID NO: 13)QIQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVG YDALDYWGQGTLVTVSS;SEQ ID No: 24 of US 2008/0025980: (SEQ ID NO: 14)QIQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTDSGESTYAEEFKGRFAFSLDTSVNTAYLQITSLNAEDTGMYFCVRVG YDALDYWGQGTLVTVSS.

In an embodiment, the targeting moiety comprises a light chaincomprising SEQ ID NO:18 of US 2008/0025980 and a heavy chain comprisingSEQ ID NO:22 of US 2008/0025980.

In an embodiment, the targeting moiety comprises AMP-514 (akaMEDI-0680).

In an embodiment, the targeting moiety comprises the PD-L2-Fc fusionprotein AMP-224, which is disclosed in WO2010/027827 and WO 2011/066342,the entire disclosures of which are hereby incorporated by reference. Insuch an embodiment, the targeting moiety may include a targeting domainwhich comprises SEQ ID NO:4 of WO2010/027827:

(SEQ ID NO: 15) LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIALRKQLCQKLYSSKDTTKRPVTTTKREV NSAI

and/or the B7-DC fusion protein which comprises SEQ ID NO:83 ofWO2010/027827:

(SEQ ID NO: 16) MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK

In an embodiment, the targeting moiety comprises the peptide AUNP 12 orany of the other peptides disclosed in US 2011/0318373 or U.S. Pat. No.8,907,053. For example, the targeting moiety may comprise AUNP 12 (i.e.,Compound 8 or SEQ ID NO:49 of US 2011/0318373) which has the sequenceof:

-   -   SNTSESFK (SNTSESF) FRVTQLAPKAQIKE (SEQ ID NO: 17)-NH2

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody1E3, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1E3 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 18) EVQLQQSGPV LVKPGASVKM SCKASGYTFT DYYMNWVKQS HGKSLEWIGNINPYNGGTTY NQKFKGKATL TVDKSSRTAY MEINSLTSED SAVYYCARGR IYDGSLDYWGQGTALTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 19) DIQMTQFPSS LCASQGGKVT VTCKASQDIN NYMAWYQHKP GKGPRLLIHYTSTLLSGIPS RFSGSGSGRD YSFSISNLEP EDIATYYCLQ YDNLWTFGGG TKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody1E8, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1E8 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 20) QVQLQQSGAE LAKPGASVRL SCKASGYTFT NYWMHWVKQR PGQGLEWIGHINPSSGFTTY NQNFKDKATL TADKSSNTAY MQLSSLTYED SAVYFCARED YDVDYWGQGTTLTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 21) DIVMTQSQKF MSTSVGDRVS VTCKASQSVD TNVAWYQQKP GQSPKALIFSASYRYSGVPD RFTGSGSGTD FTLTINSVQS EDLAEYFCQQ YNSYPYTFGS GTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody1H3, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1H3 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 22) EVQLVESGGG LVKPGGSLKL SCAASGFTFS DYGMHWVRQA PEKGLEWVAYISSGSYTIYY TDTVKGRFTI SRDNAKNTLF LQMTSLRSED TAMYYCARRG YGSFYEYYFDYWGQGTTLTV SS;

and/or light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 23) QIVLTQSPAL MSASPGEKVT MTCSASSSVS YMYWYQQKPR SSPKPWIYLTSNLASGVPAR FSGSGSGTSY SLTISSMEAE DAATYYCQQW SSNPFTFGSG TKLEIK.

In an embodiment, the targeting moiety comprises a VHH directed againstPD-1 as disclosed, for example, in U.S. Pat. No. 8,907,065 and WO2008/071447, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, the VHH s against PD-1 compriseSEQ ID NOS: 347-351 of U.S. Pat. No. 8,907,065:

SEQ ID No: 347 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 24)EVQLVESGGGLVQAGKSLRLSCAASGSIFSIHAMGWFRQAPGKEREFVAAITWSGGITYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAADR AESSWYDYWGQGTQVTVSS;SEQ ID No: 348 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 25)EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDK HQSSWYDYWGQGTQVTVSS;SEQ ID No: 349 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 26)EVQLVESGGGLVQAGGSLRLSCAASGSISSIHAMGWFRQAPGKEREFVAAITWSGGITYYADSLKGRFTISRDNAKNTGYLQMNSLKPEDTAIYYCAADR AQSSWYDYWGQGTQVTVSS;SEQ ID No: 350 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 27)EVQLVESGGGLVQAGGSLGLSCAASGSIFSINAMAWFRQAPGKEREFVALISWSGGSTYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAADR VDSNWYDYWGQGTQVTVSS;SEQ ID No: 351 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 28)EVQLVESGGGLVQAGGSLRLSCAASGRAFSSGTMGWFRRAPGKEREFVASIPWSGGRIYYADSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCAVKE RSTGWDFASWGQCTQVTVSS.

In an embodiment, the targeting moiety comprises any one of theanti-PD-1 antibodies, or fragments thereof, as disclosed inUS2011/0271358 and WO2010/036959, the entire contents of which arehereby incorporated by reference. In illustrative embodiments, theantibody or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID NOS: 25-29 of US2011/0271358:

SEQ ID No: 25 of US2011/0271358: (SEQ ID NO: 29)QVQLVQSGAELKQPGASVKMSCKASGYSFTSSWIHWVKQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDSAVYYCARWRDSSGYHAMDYWGQGTSVTVSS; SEQ ID No: 26 of US2011/0271358: (SEQ ID NO: 30)QVQLVQSGAEVKQPGASVKMSCKASGYSFTSSWIHWVKQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDTAVYY3/d10CARWRDSSGYHAMDYWGQGTSVTVSS; SEQ ID No: 27 of US2011/0271358: (SEQ ID NO:31) QVQLVQSGHEVKQPGASVKMSCKASGYSFTSSWIHWVKQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDTAVYYCARWRDSSGYHAMDYWGQGTLVTVSS; SEQ ID No: 28 of US2011/0271358: (SEQ ID NO: 32)QVQLVQSGHEVKQPGASVKMSCKASGYSFTSSWIHWVRQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDTAVYYCARWRDSSGYHAMDYWGQGTLVTVSS; SEQ ID No: 29 of US2011/0271358: (SEQ ID NO: 33)QVQLVQSGHEVKQPGASVKVSCKASGYSFTSSWIHWVRQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATITADKSTSTAYMELSSLRSEDTAVYYCARWRDSSGYHAMDYWGQGTLVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID NOS: 30-33 of US2011/0271358:

SEQ ID No: 30 of US2011/0271358: (SEQ ID NO: 34)DIVLTQSPASLTLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEEEDFATYYCQHSWEIPY TFGQGTKLEIK; SEQ IDNo: 31 of US2011/0271358: (SEQ ID NO: 35)DIVLTQSPATLSLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQHSWEIPY TFGQGTKLEIK; SEQ IDNo: 32 of US2011/0271358: (SEQ ID NO: 36)EIVLTQSPATLSLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQHSWEIPY TFGQGTKLEIK; SEQ IDNo: 33 of US2011/0271358: (SEQ ID NO: 37)DIVLTQSPATLSLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSWEIPY TFGQGTKLEIK.

In various embodiments, the present chimeric protein comprises one ormore antibodies directed against PD-1, or antibody fragments thereof,selected from TSR-042 (Tesaro, Inc.), REGN2810 (RegeneronPharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals), and BGB-A317(BeiGene Ltd.)

In various embodiments, the present chimeric protein has one or moretargeting moieties directed against PD-L1. In some embodiments, thechimeric protein has one or more targeting moieties which selectivelybind a PD-L1 polypeptide. In some embodiments, the chimeric proteincomprises one or more antibodies, antibody derivatives or formats,peptides or polypeptides, or fusion proteins that selectively bind aPD-L1 polypeptide.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyMEDI4736 (aka durvalumab), or fragments thereof. MEDI4736 is selectivefor PD-L1 and blocks the binding of PD-L1 to the PD-1 and CD80receptors. MEDI4736 and antigen-binding fragments thereof for use in themethods provided herein comprises a heavy chain and a light chain or aheavy chain variable region and a light chain variable region. Thesequence of MEDI4736 is disclosed in WO/2016/06272, the entire contentsof which are hereby incorporated by reference. In illustrativeembodiments, MEDI4736 or an antigen-binding fragment thereof for use inthe methods provided herein comprises a heavy chain comprising the aminoacid sequence of:

(SEQ ID NO: 38) EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYWMSWVRQA PGKGLEWVANIKQDGSEKYY VDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREG GWFGELAFDYWGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSGVHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEFEG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHNAKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPASIEKTI SKAKGQPREPQVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFLYSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 39) EIVLTQSPGT LSLSPGERAT LSCRASQRVS SSYLAWYQQK PGQAPRLLIYDASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSLPWTFG QGTKVEIKRTVAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSKDSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC.

In illustrative embodiments, the MEDI4736 or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:4 ofWO/2016/06272:

(SEQ ID NO: 40) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof SEQ ID NO:3 of WO/2016/06272:

(SEQ ID NO: 41) EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFG QGTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyatezolizumab (aka MPDL3280A, RG7446), or fragments thereof. Inillustrative embodiments, atezolizumab or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chaincomprising the amino acid sequence of:

(SEQ ID NO: 42) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 43) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyavelumab (aka MSB0010718C), or fragments thereof. In illustrativeembodiments, avelumab or an antigen-binding fragment thereof for use inthe methods provided herein comprises a heavy chain comprising the aminoacid sequence of:

(SEQ ID NO: 44) EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYIMMWVRQA PGKGLEWVSSIYPSGGITFY ADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARIK LGTVTTVDYWGQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 45) QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMIYDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTRV FGTGTKVTVLGQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK AGVETTKPSKQSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyBMS-936559 (aka 12A4, MDX-1105), or fragments thereof, as disclosed inUS 2013/0309250 and WO2007/005874, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments,BMS-936559 or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain variable region comprising theamino acid sequence of:

(SEQ ID NO: 46) QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 47) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3G10, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 3G10 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 48) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGFSWVRQAPGQGLEWMGWITAYNGNTNYAQKLQGRVTMTTDTSTSTVYMELRSLRSDDTAVYYCARDY FYGMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 49) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQ GTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody10A5, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 10A5 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 50) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARER IQLWFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 51) DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQ GTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody5F8, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 5F8 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 52) QVQLVQSGAEVKKPGSSVKVSCKVSGGIFSTYAINWVRQAPGQGLEWMGGIIPIFGTANHAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQ GIAAALFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 53) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFG QGTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody10H10, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 10H10 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 54) EVQLVESGGGLVQPGRSLRLSCAVSGFTFDDYVVHWVRQAPGKGLEWVSGISGNSGNIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAVPF DYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 55) DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQ GTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody1B12, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 1B12 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 56) QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSSYAISWVRQAPGQGLEWMGGIIPIFGRAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 57) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody7H1, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 7H1 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 58) QVQLVQSGAEVKKPGSSVKVSCKTSGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTTTAYMELSSLRSEDTAVYYCARKYDYVSGSPFGMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 59) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody11E6, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 11E6 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 60) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGSANYAQKFQDRVTITADESTSAAYMELSSLRSEDTAVYYCARDSSGWSRYYMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 61) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFGGG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody12B7, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 12B7 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 62) QVQLVQSGAEVKEPGSSVKVSCKASGGTFNSYAISWVRQAPGQGLEWMGGIIPLFGIAHYAQKFQGRVTITADESTNTAYMDLSSLRSEDTAVYYCARKYSYVSGSPFGMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 63) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQG TRLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody13G4, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 13G4 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 64) EVQLVESGGGLVQPGRSLRLSCAASGITFDDYGMHWVRQAPGKGLEWVSGISWNRGRIEYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGRFRYFDWFLDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 65) AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPFTFGP GTKVDIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody1E12, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1E12 or an antigen-binding fragment thereoffor use in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 66) EVKLQESGPS LVKPSQTLSL TCSVTGYSIT SDYWNWIRKF PGNKLEYVGYISYTGSTYYN PSLKSRISIT RDTSKNQYYL QLNSVTSEDT ATYYCARYGG WLSPFDYWGQGTTLTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 67) DIVMTQSHKL MSTSVGDRVS ITCKASQDVG TAVAWYQQKP GQSPKLLIYWASTRHTGVPD RFTGSGSGTD FTLTISNVQS EDLADYFCQQ DSSYPLTFGA GTKVELK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody1F4, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1F4 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 68) EVQLQESGPG LVAPSQSLSI TCTVSGFSLT TYSINWIRQP PGKGLEWLGVMWAGGGTNSN SVLKSRLIIS KDNSKSQVFL KMNSLQTDDT ARYYCARYYG NSPYYAIDYWGQGTSVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 69) DIVTTQSHKL MSTSVGDRVS ITCKASQDVG TAVAWYQQKP GQSPKLLIYWASTRHTGVPD RFTGSGSGTD FTLTISNVQS EDLADYFCQQ DSSYPLTFGA GTKVELK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2G11, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 2G11 or an antigen-binding fragment thereoffor use in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 70) EVKLQESGPS LVKPSQTLSL TCSVTGYSII SDYWNWIRKF PGNKLEYLGYISYTGSTYYN PSLKSRISIT RDTSKNQYYL QLNSVTTEDT ATYYCARRGG WLLPFDYWGQGTTLTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 71) DIVMTQSPSS LAVSVGEKVS MGCKSSQSLL YSSNQKNSLA WYQQKPGQSPKLLIDWASTR ESGVPDRFTG SGSGTDFTLT ISSVKAEDLA VYYCQQYYGY PLTFGAGTKL ELK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3B6, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 3B6 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 72) EVKLQESGPS LVKPGASVKL SCKASGYTFT SYDINWVKQR PGQGLEWIGWIFPRDNNTKY NENFKGKATL TVDTSSTTAY MELHSLTSED SAVYFCTKEN WVGDFDYWGQGTTLTLSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 73) DIVMTQSPAI MSASPGEKVT MTCSASSSIR YMHWYQQKPG TSPKRWISDTSKLTSGVPAR FSGSGSGTSY ALTISSMEAE DAATYYCHQR SSYPWTFGGG TKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3D10, or fragments thereof, as disclosed in US 2014/0044738 andWO2012/145493, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 3D10 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 74) EVQLQQSGPD LVTPGASVRI SCQASGYTFP DYYMNWVKQS HGKSLEWIGDIDPNYGGTTY NQKFKGKAIL TVDRSSSTAY MELRSLTSED SAVYYCARGA LTDWGQGTSL TVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 75) QIVLSQSPAI LSASPGEKVT MTCRASSSVS YIYWFQQKPG SSPKPWIYATFNLASGVPAR FSGSGSGTSY SLTISRVETE DAATYYCQQW SNNPLTFGAG TKLELK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in US2011/0271358 and WO2010/036959, theentire contents of which are hereby incorporated by reference. Inillustrative embodiments, the antibody or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chaincomprising an amino acid sequence selected from SEQ ID Nos: 34-38 ofUS2011/0271358:

SEQ ID No: 34 of US2011/0271358: (SEQ ID NO: 76)EVQLVQSGPELKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDSAVYYCARQA WGYPWGQGTLVTVSS; SEQID No: 35 of US2011/0271358: (SEQ ID NO: 77)EVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCARQA WGYPWGQGTLVTVSS; SEQID No: 36 of US2011/0271358: (SEQ ID NO: 78)EVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCARQA WGYPWGQGTLVTVSS; SEQID No: 37 of US2011/0271358: (SEQ ID NO: 79)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCARQA WGYPWGQGTLVTVSS; SEQID No: 38 of US2011/0271358: (SEQ ID NO: 80)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATITSDKSTSTAYMELSSLRSEDTAVYYCARQA WGYPWGQGTLVTVSS;

SEQ ID No: 39 of US2011/0271358: (SEQ ID NO: 81)DIVLTQSPASLALSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEEEDAAMYFCQQSRRVPY TEGQGTKLEIK;SEQ ID No: 40 of US2011/0271358: (SEQ ID NO: 82)DIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAAMYFCQQSRRVPY TFGQGTKLEIK;SEQ ID No: 41 of US2011/0271358: (SEQ ID NO: 83)EIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAAMYFCQQSRRVPY TFGQGTKLEIK;SEQ ID No: 42 of US2011/0271358: (SEQ ID NO: 84)DIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAATYFCQQSRRVPY TFGQGTKLEIK.

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 39-42 of US2011/02713558:

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.7A4, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.7A4 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 2 of WO 2011/066389: (SEQ ID NO: 85)EVQLVESGGGLVKPGGSLRLSCAASGFTESTYSMNWVRQAPGKGLEWVSSISSSGDYIYYADSVKGRETISRDNAKNSLFLQMNSLKAEDTAVYYCARDL VTSMVAFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 7 of WO 2011/066389: (SEQ ID NO: 86)SYELTQPPSVSVSPGQAARITCSGDALPQKYVFWYQQKSGQAPVLVIYEDSKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDRSGNHRVFG GGTRLTVL.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.9D10, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.9D10 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 12 of WO 2011/066389: (SEQ ID NO: 87)EVQLVESGGGLVQPGGSLRLSCAASGFTESSYWMSWVRQAPGKGLEWVANIKQDGGEQYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDW NYGYYDMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 17 of WO 2011/066389: (SEQ ID NO: 88)EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWFQQKPGQAPRLLIFGTSSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSIFTFG PGTKVDIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.14H9, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.14H9 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 22 of WO 2011/066389: (SEQ ID NO: 89)EVQLVESGGGLVQPGGSLRLSCAASGETFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 27 of WO 2011/066389: (SEQ ID NO: 90)EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFG QGTEVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.20A8, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.20A8 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 32 of WO 2011/066389: (SEQ ID NO: 91)EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAIRGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLHYDSSGYLDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 37 of WO 2011/066389: (SEQ ID NO: 92)DIQMTQSPSSVSASVGDRVTITCRASQGIRSWLAWYQQKPGKAPKLLIYAISRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGG GTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3.15G8, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 3.15G8 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 42 of WO 2011/066389: (SEQ ID NO: 93)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGGEKYYVDSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARVQ LYSDYFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 47 of WO 2011/066389: (SEQ ID NO: 94)DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKSGKAPKLLIYAASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSHSLPPTFGQ GTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3.18G1, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 3.18G1 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 52 of WO 2011/066389: (SEQ ID NO: 95)EVQLLESGGDLVQPGGSLRLSCAASGFTFNSYAMSWVRQAPGKGLEWVSTISGSGGFTFSADSVKGRFTISRDNSKNTLFLQMNSLRVEDSAVYSCAKVLVGFNNGCWDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 57 of WO 2011/066389: (SEQ ID NO: 96)SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSNDHVVFG GGTKLTVL.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.7A4OPT, or fragments thereof, as disclosed in WO 2011/066389, U.S.Pat. No. 8,779,108, and US2014/0356353, and US2014/0356353, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 2.7A4OPT or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chainvariable region comprising the amino acid sequence of:

SEQ ID No: 62 of WO 2011/066389: (SEQ ID NO: 97)EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYSMNWVRQAPGKGLEWVSSISSSGDYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDL VTSMVAFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 67 of WO 2011/066389: (SEQ ID NO: 98)SYELTQPPSVSVSPGQTARITCSGDALPQKYVFWYQQKSGQAPVLVIYEDSKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDRSGNHRVFG GGTKLTVL.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.14H9OPT, or fragments thereof, as disclosed in WO 2011/066389, U.S.Pat. No. 8,779,108, and US2014/0356353, the entire disclosures of whichare hereby incorporated by reference. In illustrative embodiments,2.14H9OPT or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain variable region comprising theamino acid sequence of:

SEQ ID No: 72 of WO 2011/066389: (SEQ ID NO: 99)EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 77 of WO 2011/066389: (SEQ ID NO: 100)EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFG QGTKVEIK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO2016/061142, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 18, 30, 38, 46, 50, 54, 62, 70, and78 of WO2016/061142:

SEQ ID No: 18 of WO2016/061142: (SEQ ID NO: 101)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMYWVRQATGQGLEWMGRIDPNSGSTKYNEKFKNRFTISRDDSKNTAYLQMNSLKTEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 30 of WO2016/061142: (SEQ ID NO: 102)EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWVRQATGQGLEWMGRIDPNSGSTKYNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 38 of WO2016/061142: (SEQ ID NO: 103)EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMYWVRQAPGQGLEWMGRIDPNSGSTKYNEKFKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 46 of WO2016/061142: (SEQ ID NO: 104)EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWIRQSPSRGLEWLGRIDPNSGSTKYNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 50 of WO2016/061142: (SEQ ID NO: 105)EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMYWIRQPPGKGLEWIGRIDPNSGSTKYNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 54 of WO2016/061142: (SEQ ID NO: 106)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMYWIRQSPSRGLEWLGRIDPNSGSTKYNEKFKNRFTISRDDSKNTAYLQMNSLKTEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 62 of WO2016/061142: (SEQ ID NO: 107)EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMYWVRQARGQRLEWIGRIDPNSGSTKYNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 70 of WO2016/061142: (SEQ ID NO: 108)QITLKESGPTLVKPTQTLTLTCTFSGYTFTSYWMYWVRQAPGKGLEWVSRIDPNSGSTKYNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 78 of WO2016/061142: (SEQ ID NO: 109)EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWVRQARGQRLEWIGRIDPNSGSTKYNEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 22, 26, 34, 42, 58, 66, 74, 82, and 86 of WO2016/061142:

SEQ ID No: 22 of WO2016/061142: (SEQ ID NO: 110)DIVMTQTPLSLPVTPGEPASISCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:26 of WO2016/061142: (SEQ ID NO: 111)DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:34 of WO2016/061142: (SEQ ID NO: 112)EIVLTQSPDFQSVTPKEKVTITCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:42 of WO2016/061142: (SEQ ID NO: 113)EIVLTQSPDFQSVTPKEKVTITCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNSYPLTFGQ GTKVEIK. SEQ ID No:58 of WO2016/061142: (SEQ ID NO: 114)EIVLTQSPATLSLSPGERATLSCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:66 of WO2016/061142: (SEQ ID NO: 115)DVVMTQSPLSLPVTLGQPASISCKASQDVGTAVAWYQQKPGQAPRLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:74 of WO2016/061142: (SEQ ID NO: 116)DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGQAPRLLIYWASTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:82 of WO2016/061142: (SEQ ID NO: 117)AIQLTQSPSSLSASVGDRVTITCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:86 of WO2016/061142: (SEQ ID NO: 118)EIVLTQSPDFQSVTPKEKVTITCKASQDVGTAVAWYQQKPGQAPRLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPLTFGQ GTKVEIK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO2016/022630, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38,42, and 46 of WO2016/022630:

SEQ ID No: 2 of WO2016/022630: (SEQ ID NO: 119)EVKLVESGGGLVKPGGSLKLSCAASGFIFRSYGMSWVRQTPEKRLEWVASISSGGSTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYDCARGYD SGFAYWGQGTLVTVSE; SEQID No: 6 of WO2016/022630: (SEQ ID NO: 120)EVKLVESGGGLVKPGGSLKLSCAASGFTFRSYGMSWVRQTPEKRLEWVASISSGGTTYYPDSVKGRFIISRDNARNILYLQMSSLRSEDTAMYYCAKGYD SGFAYWGQGTLVIVSA; SEQID No: 10 of WO2016/022630: (SEQ ID NO: 121)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWRGVTTDYNAAFMSRLTITKDNSKSQVFFKMNSLQANDTAIYYCARLGF YAMDYWGQGTSVTVSS; SEQID No: 14 of WO2016/022630: (SEQ ID NO: 122)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGVTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCARLGF YAMDYWGQGTSVTVSS; SEQID No: 18 of WO2016/022630: (SEQ ID NO: 123)EVKLFESGGGLVQPGGSLKLSCVASGFDFSTYWMHWVRQAPGQGLEWIGQINPDSTTINYAPSLKDRFIISRDNAKNTLFLQMSKVRSEDTALYYCAKPG DYGYDFDCWGQGTTLTVSS;SEQ ID No: 22 of WO2016/022630: (SEQ ID NO: 124)EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARSLL WFSTGFAYWGQGTLVTVSA;SEQ ID No: 26 of WO2016/022630: (SEQ ID NO: 125)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGITDYNAAFKSRLSISKDNSKSQVFFKMNSLQANDTAIYFCARLGF YAMDYWGQGTSVTVSS; SEQID No: 30 of WO2016/022630: (SEQ ID NO: 126)EVKLVESGGGLVKPGGSLKLSCAASGFTFRSYGMSWARQIPEKRLEWVASISSGGTTYYLGSVQGRFTISRDNARNILYLQMSSLRSEDTAMYYCARGYD AGFAYWGQGTLVSVSE; SEQID No: 34 of WO2016/022630: (SEQ ID NO: 127)EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWTWIRKFPGNKLEYMGYISYTGSTYYNPSLKSRISISRDTSKSQYYLQLNSVTTEDTATYYCARQRD WLGFAYWGQGTLVTVSA;SEQ ID No: 38 of WO2016/022630: (SEQ ID NO: 128)EEKLVESGGGLVKPGGSLKLSCAASGFSFSSYGMSWVRQTPEKRLEWVASISSGGSIYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARGYD AGFAFWGQGTLVTASA; SEQID No: 42 of WO2016/022630: (SEQ ID NO: 129)QITLKESGPTLVKPTQTLTLTCTVSGFSLSTYGVHWIRQPPGKALEWLGVIWRGVTTDYNAAFMSRLTITKDNSKNQVVLTMNNMDPVDTATYYCARLGF YAMDYWGQGTLVTVSS; SEQID No: 46 of WO2016/022630: (SEQ ID NO: 130)EVQLVESGGGLVKPGGSLRLSCAASGFIFRSYGMSWVRQAPGKGLEWVASISSGGSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYDCARGYD SGFAYWGQGTLVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 ofWO2016/022630:

SEQ ID No: 4 of WO2016/022630: (SEQ ID NO: 131)DIVLTQSPASLAVSLGQRATISCRASQSVSTSSSSFMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPY TFGGGTKLEIKR; SEQ IDNo: 8 of WO2016/022630: (SEQ ID NO: 132)DIVLTQSPPSLAVSLGQRATISCRASQSVSTSSSSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPY TFGGGTKLEIK; SEQ IDNo: 12 of WO2016/022630: (SEQ ID NO: 133)SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYAANRYTGVPDRFTGSGYGTDFTFTISIVQAEDLAVYFCQQDYTSPYTFGG GTKLEIK; SEQ ID No:16 of WO2016/022630: (SEQ ID NO: 134)SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVGWYQQKPGQSPKLLIYYASNRYSGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYTSPYTFGG GTKLEIK; SEQ ID No:20 of WO2016/022630: (SEQ ID NO: 135)DVLMTQTPLYLPVSLGDQASISCRSSQIIVHSNANTYLEWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP YTFGGGTKLEIK; SEQ IDNo: 24 of WO2016/022630: (SEQ ID NO: 136)QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWNQQKPGSSPKVWIYNTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWRSYPPTLG AGTKLELK; SEQ ID No:28 of WO2016/022630: (SEQ ID NO: 137)QIVLTQSPAIMSASPGEKVTMTCSANSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMGAEDAATYYCQQWSSNPWTFGGG TKLEIK; SEQ ID No: 32of WO2016/022630: (SEQ ID NO: 138)DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQNSWEIPY TFGGGTKLEIK; SEQ IDNo: 36 of WO2016/022630: (SEQ ID NO: 139)DIVMTQTPSSLAVSLGEKVTMSCKSSQSLLYSSNQKNSLAWYQQKPGQSPKLLIYWASNRESGVPDRFTGSSSGTDFTLTISSVKAEDLAVYYCQQYYSY PLTFGAGTKLELK; SEQ IDNo: 40 of WO2016/022630: (SEQ ID NO: 140)DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYVHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPY TFGGGTKLEIK; SEQ IDNo: 44 of WO2016/022630: (SEQ ID NO: 141)DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYAANRYTGVPDRFSGSGYGTDFTFTISSLQPEDIATYFCQQDYTSPYTFGQ GTKLEIK; SEQ ID No:48 of WO2016/022630: (SEQ ID NO: 142)DIVLTQSPASLAVSPGQRATITCRASQSVSTSSSSFMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSWEIPY TFGQGTKLEIK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO2015/112900, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 38, 50, 82, and 86 of WO 2015/112900:

SEQ ID No: 38 of WO2015/112900: (SEQ ID NO: 143)EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQATGQGLEWMGNIYPGTGGSNFDEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;SEQ ID No: 50 of WO 2015/112900: (SEQ ID NO: 144)EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWIRQSPSRGLEWLGNIYPGTGGSNFDEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;SEQ ID No: 82 of WO 2015/112900: (SEQ ID NO: 145)QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWIRQSPSRGLEWLGNIYPGTGGSNFDEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;SEQ ID No: 86 of WO 2015/112900: (SEQ ID NO: 146)EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQAPGQGLEWMGNIYPGTGGSNFDEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 42, 46, 54, 58, 62, 66, 70, 74, and 78 of WO 2015/112900:

SEQ ID No: 42 of WO2015/112900: (SEQ ID NO: 147)EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 46 of WO 2015/112900: (SEQ ID NO: 148)DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 54 of WO 2015/112900: (SEQ ID NO: 149)EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 58 of WO 2015/112900: (SEQ ID NO: 150)DIVMTQTPLSLPVTPGEPASISCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 62 of WO 2015/112900: (SEQ ID NO: 151)EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 66 of WO 2015/112900: (SEQ ID NO: 152)EIVLTQSPDFQSVTPKEKVTITCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 70 of WO 2015/112900: (SEQ ID NO: 153)EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 74 of WO 2015/112900: (SEQ ID NO: 154)DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSGNQKNFLTWYLQKPGQSPQLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 78 of WO 2015/112900: (SEQ ID NO: 155)DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO 2010/077634 and U.S. Pat. No.8,217,149, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, the anti-PD-L1 antibody or anantigen-binding fragment thereof for use in the methods provided hereincomprises a heavy chain region comprising the amino acid sequence of:

SEQ ID No: 20 of WO 2010/077634: (SEQ ID NO: 156)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH WPGGFDYWGQGTLVTVSA;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 21 of WO 2010/077634: (SEQ ID NO: 157)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies obtainable from the hybridoma accessible underCNCM deposit numbers CNCM I-4122, CNCM I-4080 and CNCM I-4081 asdisclosed in US 20120039906, the entire disclosures of which are herebyincorporated by reference.

In an embodiment, the targeting moiety comprises a VHH directed againstPD-L1 as disclosed, for example, in U.S. Pat. No. 8,907,065 and WO2008/071447, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, the VHHs against PD-L1 compriseSEQ ID NOS: 394-399 of U.S. Pat. No. 8,907,065:

SEQ ID No: 394 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 158)EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREWASSISSSDGSTYYADSVKGRFTISRDNAKNTVFLQMNSLKPEDTAVYSCAASQAPITIATMMKPFYDYWGQGTQVTVSS; SEQ ID No: 395 of U.S. Pat. No. 8,907,065:(SEQ ID NO: 159) EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAKCWFRQAPGKEREWVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAARHGGPLTVEYFFDYWGQGTQVTVSS; SEQ ID No: 396 of U.S. Pat. No. 8,907,065: (SEQID NO: 160) EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYAIGWFRQAPGKAREGVSCISGGDNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATGGWKYCSGYDPEYIYWGQGTQVTVSS; SEQ ID No: 397 of U.S. Pat. No. 8,907,065:(SEQ ID NO: 161) EVQLVESGGGLVQAGGSLRLSCAASGSTFSQYDVGWYRQAPGKQRELVAFSSSGGRTIYPDSVKGRFTFSRDNTKNTVYLQMTSLKPEDTAVYYCKIDWY LNSYWGQGTQVTVSS; SEQID No: 398 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 162)EVQLVESGGGLVQAGGSLRLSCAASGVDASNSAMGWYRQAPGKQREWVARITGGGLIAYTDSVKGRFTISRDNAKSTVYLQMNSLEPEDTAVYYCNTINS RDGWGQGTQVTVSS; SEQID No: 399 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 163)EVQLVESGGGLVQAGGSLTISCAASGITFSDSIVSWYRRARGKQREWVAGISNGGTTKYAESVLGRFTISRDNAKNNVYLQMNGLNPEDTAVYLCKVRQY WGQGTQVTVSS.

In various embodiments, the present chimeric protein has one or moretargeting moieties directed against PD-L2. In some embodiments, thechimeric protein has one or more targeting moieties which selectivelybind a PD-L2 polypeptide. In some embodiments, the chimeric proteincomprises one or more antibodies, antibody derivatives or formats,peptides or polypeptides, or fusion proteins that selectively bind aPD-L2 polypeptide.

In an embodiment, the targeting moiety comprises a VHH directed againstPD-L2 as disclosed, for example, in U.S. Pat. No. 8,907,065 and WO2008/071447, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, the VHHs against PD-1 compriseSEQ ID Nos: 449-455 of U.S. Pat. No. 8,907,065:

SEQ ID No: 449 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 164)EVQLVESGGGLVQAGGSLRLSCAASESTVLINAMGWYRQAPGKQRELVASISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADVYPQDYGLGYVEGKVYYGHDYWGTGTLVTVSS; SEQ ID No: 450 of U.S. Pat. No.8,907,065: (SEQ ID NO: 165)EVQLVESGGGLVQAGGSLRLSCAASGSTFSNYVSNYAMGWGRQAPGTQRELVASISNGDTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCF EHQVAGLTWGQGTQVTVSS;SEQ ID No: 451 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 166)EVQLVESGGGLVQAGGSLRLSCVASGXALKIXVMGWYRQAPGKQRELVAAITSGGRTNYSDSVKGRFTISGDNAXNTVYLQMNSLKSEDTAVYYCREWNS GYPPVDYWGQGTQVTVSS;SEQ ID No: 452 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 167)EVQLVESGGGLVQAGGSLRLSCAASGRTFSSGTMGWFRRAPGKEREFVASIPWSGGRTYYADSVKDRFTISRDNAQNTVFLQMNSLKPEDTAVYYCAFKE RSTGWDFASWGQGIQVTVSS;SEQ ID No: 453 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 168)EVQLVESGGGLVQTGGSLRLSCAASGFTLDYYGIGWFRQAPGKEREGVSFISGSDGSTYYAESVKGRFTISRDKAKNTVYLQMNSLKPEDTAVYYCAADPWGPPSIATMTSYEYKHWGQGTQVTVSS; SEQ ID No: 454 of U.S. Pat. No. 8,907,065:(SEQ ID NO: 169) EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMIWLRRAPGKGFEWVSTIDKDGNTNYVDSVKGRFAVSRDNTKNTLYLQMNSLKPEDTAMYYCTKHGS SARGQGTRVTVSS; SEQ IDNo: 455 of U.S. Pat. No. 8,907,065: (SEQ ID NO: 170)EVQLVESGGGLVEPGGSLRLSCVASGFTFSSYDMSWVRQAPGKGLEWVSTINSGGGITYRGSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCENGGS SYRRGQGTQVTVSS.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L2 antibodies disclosed in US2011/0271358 and WO2010/036959, theentire contents of which are hereby incorporated by reference. Inillustrative embodiments, the antibody or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chaincomprising an amino acid sequence selected from SEQ ID Nos: 43-47 ofUS2011/0271358:

SEQ ID No: 43 of US2011/0271358: (SEQ ID NO: 171)QVQLVQSGAELKKPGASVKMSCKASGYTFTGYTMHWVKQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDSAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 44 of US2011/0271358: (SEQ ID NO: 172)QVQLVQSGAEVKKPGASVKMSCKASGYTFTGYTMHWVKQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 45 of US2011/0271358: (SEQ ID NO: 173)QVQLVQSGAEVKKPGASVKMSCKASGYTFTGYTMHWVRQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 46 of US2011/0271358: (SEQ ID NO: 174)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMHWVRQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 47 of US2011/0271358: (SEQ ID NO: 175)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMHWVRQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTITADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 48-51 of US2011/0271358:

SEQ ID No: 48 of US2011/0271358: (SEQ ID NO: 176)DIVMTQSPASLTVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTEGQGTKLEIK;SEQ ID No: 49 of US2011/0271358: (SEQ ID NO: 177)DIVMTQSPASLSVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTFGQGTKLEIK;SEQ ID No: 50 of US2011/0271358: (SEQ ID NO: 178)DIVMTQSPAFLSVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTFGQGTKLEIK;SEQ ID No: 51 of US2011/0271358: (SEQ ID NO: 179)DIVMTQSPAFLSVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTFGQGTKLEIK.

In various embodiments, the targeting moieties of the invention maycomprise a sequence that targets PD-1, PD-L1, and/or PD-L2 which is atleast about 60%, at least about 61%, at least about 62%, at least about63%, at least about 64%, at least about 65%, at least about 66%, atleast about 67%, at least about 68%, at least about 69%, at least about70%, at least about 71%, at least about 72%, at least about 73%, atleast about 74%, at least about 75%, at least about 76%, at least about77%, at least about 78%, at least about 79%, at least about 80%, atleast about 81%, at least about 82%, at least about 83%, at least about84%, at least about 85%, at least about 86%, at least about 87%, atleast about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or 100% identical to any of the sequencesdisclosed herein (e.g. about 60%, or about 61%, or about 62%, or about63%, or about 64%, or about 65%, or about 66%, or about 67%, or about68%, or about 69%, or about 70%, or about 71%, or about 72%, or about73%, or about 74%, or about 75%, or about 76%, or about 77%, or about78%, or about 79%, or about 80%, or about 81%, or about 82%, or about83%, or about 84%, or about 85%, or about 86%, or about 87%, or about88%, or about 89%, or about 90%, or about 91%, or about 92%, or about93%, or about 94%, or about 95%, or about 96%, or about 97%, or about98%, about 99% or about 100% sequence identity with any of the sequencesdisclosed herein).

In various embodiments, the targeting moieties of the invention maycomprise any combination of heavy chain, light chain, heavy chainvariable region, light chain variable region, complementaritydetermining region (CDR), and framework region sequences that targetPD-1, PD-L1, and/or PD-L2 as disclosed herein.

Additional antibodies, antibody derivatives or formats, peptides orpolypeptides, or fusion proteins that selectively bind or target PD-1,PD-L1 and/or PD-L2 are disclosed in WO 2011/066389, US 2008/0025980, US2013/0034559, U.S. Pat. No. 8,779,108, US 2014/0356353, U.S. Pat. No.8,609,089, US 2010/028330, US 2012/0114649, WO 2010/027827, WO2011,/066342, U.S. Pat. No. 8,907,065, WO 2016/062722, WO 2009/101611,WO2010/027827, WO 2011/066342, WO 2007/005874, WO 2001/014556,US2011/0271358, WO 2010/036959, WO 2010/077634, U.S. Pat. No. 8,217,149,US 2012/0039906, WO 2012/145493, US 2011/0318373, U.S. Pat. No.8,779,108, US 20140044738, WO 2009/089149, WO 2007/00587, WO 2016061142,WO 2016,02263, WO 2010/077634, and WO 2015/112900, the entiredisclosures of which are hereby incorporated by reference.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a checkpoint marker on a T cell, for example,PD-1 and (ii) a targeting moiety directed against a tumor cell, forexample, PD-L1 or PD-L2, along with any of the modified (e.g. mutant)signaling agents described herein (e.g., modified IFN-β). In anembodiment, the present chimeric protein has a targeting moiety directedagainst PD-1 on T cells and a second targeting moiety directed againstPD-L1 on tumor cells. In another embodiment, the present chimericprotein has a targeting moiety directed against PD-1 on T cells and asecond targeting moiety directed against PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a T cell, for example, mediated by targeting toCD8 and (ii) a targeting moiety is directed against a tumor cell, alongwith any of the modified (e.g. mutant) signaling agents described herein(e.g., modified IFN-β). In an embodiment, the present chimeric proteinhas a targeting moiety directed against CD8 on T cells and a secondtargeting moiety directed against PD-L1 or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a T cell, for example, mediated by targeting toCD4 and (ii) a targeting moiety is directed against a tumor cell, alongwith any of the modified (e.g. mutant) signaling agents described herein(e.g., modified IFN-β). In an embodiment, the present chimeric proteinhas a targeting moiety directed against CD4 on T cells and a secondtargeting moiety directed against PD-L1 or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a T cell, for example, mediated by targeting toCD3, CXCR3, CCR4, CCR9, CD70, CD103, or one or more immune checkpointmarkers and (ii) a targeting moiety is directed against a tumor cell,along with any of the modified (e.g. mutant) signaling agents describedherein (e.g., modified IFN-β). In an embodiment, the present chimericprotein has a targeting moiety directed against CD3 on T cells and asecond targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a T cell, for example, mediated by targeting toPD-1 and (ii) a targeting moiety is directed against a tumor cell, alongwith any of the modified (e.g. mutant) signaling agents described herein(e.g., modified IFN-β).

By way of non-limiting example, in various embodiments, the presentchimeric protein has (i) a targeting moiety directed against a B cell,for example, mediated by targeting to CD10, CD19, CD20, CD21, CD22,CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75,CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86,CD89, CD98, CD126, CD127, CDw130, CD138, or CDw150; and (ii) a targetingmoiety is directed against a tumor cell, along with any of the modified(e.g. mutant) signaling agents described herein (e.g., modified IFN-β).

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a B cell, for example, mediated by targeting toCD19, CD20 or CD70 and (ii) a targeting moiety is directed against atumor cell, along with any of the modified (e.g. mutant) signalingagents described herein (e.g., modified IFN-β).

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a B cell, for example, mediated by targeting toCD20 and (ii) a targeting moiety is directed against a tumor cell, alongwith any of the modified (e.g. mutant) signaling agents described herein(e.g., modified IFN-β). In an embodiment, the present chimeric proteinhas a targeting moiety directed against CD20 on B cells and a secondtargeting moiety directed against PD-L1 or PD-L2 on tumor cells.

By way of non-limiting example, in various embodiments, the presentchimeric protein has (i) a targeting moiety directed against a NK cell,for example, mediated by targeting to 2B4/SLAMF4, KIR2DS4, CD155/PVR,KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7,LMIR3/CD300LF, DNAM-1, LMIR5/CD300LB, Fc-epsilon RII, LMIR6/CD300LE,Fc-γ RI/CD64, MICA, Fc-γ RIIB/CD32b, MICB, Fc-γ RIIC/CD32c, MULT-1, Fc-γRIIA/CD32a, Nectin-2/CD112, Fc-γ RIII/CD16, NKG2A, FcRH1/IRTA5, NKG2C,FcRH2/IRTA4, NKG2D, FcRH4/IRTA1, NKp30, FcRH5/IRTA2, NKp44,Fc-Receptor-like 3/CD16-2, NKp46/NCR1, NKp80/KLRF1, NTB-A/SLAMF6, Rae-1,Rae-1 α, Rae-1 β, Rae-1 delta, H60, Rae-1 epsilon, ILT2/CD85j, Rae-1 γ,ILT3/CD85k, TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3, KIR/CD158,TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4/CD158d, orULBP-3; and (ii) a targeting moiety is directed against a tumor cell,along with any of the modified (e.g. mutant) signaling agents describedherein (e.g., modified IFN-β).

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a NK cell, for example, mediated by targeting toKir1alpha, DNAM-1 or CD64 and (ii) a targeting moiety is directedagainst a tumor cell, along with any of the modified (e.g. mutant)signaling agents described herein (e.g., modified IFN-β).

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a NK cell, for example, mediated by targeting toKIR1 and (ii) a targeting moiety is directed against a tumor cell, alongwith any of the modified (e.g. mutant) signaling agents described herein(e.g., modified IFN-β). In an embodiment, the present chimeric proteinhas a targeting moiety directed against KIR1 on NK cells and a secondtargeting moiety directed against PD-L1 or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a NK cell, for example, mediated by targeting toTIGIT or KIR1 and (ii) a targeting moiety is directed against a tumorcell, along with any of the modified (e.g. mutant) signaling agentsdescribed herein (e.g., modified IFN-β). In an embodiment, the presentchimeric protein has a targeting moiety directed against TIGIT on NKcells and a second targeting moiety directed against PD-L1 or PD-L2 ontumor cells.

By way of non-limiting example, in various embodiments, the presentchimeric protein has (i) a targeting moiety directed against a dendriticcell, for example, mediated by targeting to CLEC-9A, XCR1, RANK,CD36/SRB3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-A1/MSR, CD5L, SREC-1,CL-PI/COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2,TLR6, TLR3, TLR9, 4-IBB Ligand/TNFSF9, IL-12/IL-23 p40,4-Amino-1,8-naphthalimide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k,8-oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, lutegrin α 4/CD49d, Aag,Integrin β 2/CD18, AMICA, Langerin, B7-2/CD86, Leukotriene B4 RI, B7-H3,LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, Clq R1/CD93, LMIR3/CD300LF,CCR6, LMIR5/CD300LB CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4-a,CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5,MMR, CD97, NCAMLI, CD2F-10/SLAMF9, Osteoactivin GPNMB, Chern 23, PD-L2,CLEC-1, RP105, CLEC-2, Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33,DC-SIGN, Siglec-5, DC-SIGNR/CD299, Siglec-6, DCAR, Siglec-7,DCIR/CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1/CLEC7A, Siglec-F,Dectin-2/CLEC6A, SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM,EMMPRIN/CD147, TCCR/WSX-1, Fc-γ R1/CD64, TLR3, Fc-γ RIIB/CD32b, TREM-1,Fc-γ RIIC/CD32c, TREM-2, Fc-γ RIIA/CD32a, TREM-3, Fc-γ RIII/CD16,TREML1/TLT-1, ICAM-2/CD102, or Vanilloid R1; and (ii) a targeting moietythat is directed against a tumor cell or immune cell, along with any ofthe modified (e.g. mutant) signaling agents described herein (e.g.,modified IFN-β).

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a dendritic cell, for example, mediated bytargeting to CLEC-9A, DC-SIGN, CD64, CLEC4A, or DEC205 and (ii) atargeting moiety is directed against a tumor cell, along with any of themodified (e.g. mutant) signaling agents described herein (e.g., modifiedIFN-β). In an embodiment, the present chimeric protein has a targetingmoiety directed against CLEC9A on dendritic cells and a second targetingmoiety directed against PD-L1 or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a dendritic cell, for example, mediated bytargeting to CLEC9A and (ii) a targeting moiety is directed against atumor cell, along with any of the modified (e.g. mutant) signalingagents described herein (e.g., modified IFN-β). In an embodiment, thepresent chimeric protein has a targeting moiety directed against CLEC9Aon dendritic cells and a second targeting moiety directed against PD-L1or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a dendritic cell, for example, mediated bytargeting to XCR1 and (ii) a targeting moiety is directed against atumor cell, along with any of the modified (e.g. mutant) signalingagents described herein (e.g., modified IFN-β). In an embodiment, thepresent chimeric protein has a targeting moiety directed against XCR1 ondendritic cells and a second targeting moiety directed against PD-L1 orPD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a dendritic cell, for example, mediated bytargeting to RANK and (ii) a targeting moiety is directed against atumor cell, along with any of the modified (e.g. mutant) signalingagents described herein (e.g., modified IFN-β). In an embodiment, thepresent chimeric protein has a targeting moiety directed against RANK ondendritic cells and a second targeting moiety directed against PD-L1 orPD-L2 on tumor cells.

By way of non-limiting example, in various embodiments, the presentchimeric protein has (i) a targeting moiety directed against amonocyte/macrophage, for example, mediated by targeting to SIRP1a,B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, Common β Chain, Integrin α4/CD49d, BLAME/SLAMF8, Integrin α X/CDIIc, CCL6/C10, Integrin β 2/CD18,CD155/PVR, Integrin β 3/CD61, CD31/PECAM-1, Latexin, CD36/SR-B3,Leukotriene B4 R1, CD40/TNFRSF5, LIMPIIISR-B2, CD43, LMIR1/CD300A, CD45,LMIR2/CD300c, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97,LMIR6/CD300LE, CD163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1,ECF-L, MD-2, EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB,Fc-γ RI/CD64, Osteopontin, Fc-γ RIIB/CD32b, PD-L2, Fc-γ RIIC/CD32c,Siglec-3/CD33, Fc-γ RIIA/CD32a, SIGNR1/CD209, Fc-γ RIII/CD16, SLAM,GM-CSF R α, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-γ RI, TLR4, IFN-gannnaR2, TREM-I, IL-I RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k,TREML1/TLT-1, 2B4/SLAMF 4, IL-10 R α, ALCAM, IL-10 R β,AminopeptidaseN/ANPEP, ILT2/CD85j, Common β Chain, ILT3/CD85k, ClqR1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, CD206, Integrin α 4/CD49d,CCR5, Integrin α M/CDII b, CCR8, Integrin α X/CDIIc, CD155/PVR, Integrinβ 2/CD18, CD14, Integrin β 3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45,Leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97,LMIR2/CD300c, CD163, LMIR3/CD300LF, Coagulation Factor III/TissueFactor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1,CXCR6, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR,Endoglin/CD105, NCAM-L1, Fc-γ RI/CD64, PSGL-1, Fc-γ RIIIICD16, RP105,G-CSF R, L-Selectin, GM-CSF R α, Siglec-3/CD33, HVEM/TNFRSF14, SLAM,ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-I, IL-6 R, TREM-2,CXCRI/IL-8 RA, TREM-3, or TREMLI/TLT-1; and (ii) a targeting moiety isdirected against a tumor cell, along with any of the modified (e.g.mutant) signaling agents described herein (e.g., modified IFN-β).

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a monocyte/macrophage, for example, mediated bytargeting to B7-H1, CD31/PECAM-1, CD163, CCR2, or Macrophage MannoseReceptor CD206 and (ii) a targeting moiety is directed against a tumorcell, along with any of the modified (e.g. mutant) signaling agentsdescribed herein (e.g., modified IFN-β).

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a monocyte/macrophage, for example, mediated bytargeting to SIRP1a and (ii) a targeting moiety is directed against atumor cell, along with any of the modified (e.g. mutant) signalingagents described herein (e.g., modified IFN-β). In an embodiment, thepresent chimeric protein has a targeting moiety directed against SIRP1aon macrophage cells and a second targeting moiety directed against PD-L1or PD-L2 on tumor cells.

In various embodiments, the present chimeric protein has one or moretargeting moieties directed against a checkpoint marker, e.g. one ormore of PD-1/PD-L1 or PD-L2, CD28/CD80 or CD86, CTLA4/CD80 or CD86,ICOS/ICOSL or B7RP1, BTLA/HVEM, KIR, LAG3, CD137/CD137L, OX40/OX40L,CD27, CD40L, TIM3/Gal9, and A2aR.

In some embodiments, the present chimeric protein comprises two or moretargeting moieties directed to the same or different immune cells. Insome embodiments, the present chimeric protein has (i) one or moretargeting moieties directed against an immune cell selected from a Tcell, a B cell, a dendritic cell, a macrophage, a NK cell, or subsetsthereof and (ii) one or more targeting moieties directed against eitherthe same or another immune cell selected from a T cell, a B cell, adendritic cell, a macrophage, a NK cell, or subsets thereof, along withany of the modified (e.g. mutant) signaling agents described herein(e.g., modified IFN-β).

In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a T cell and one or more targetingmoieties directed against the same or another T cell. In one embodiment,the present chimeric protein comprises one or more targeting moietiesdirected against a T cell and one or more targeting moieties directedagainst a B cell. In one embodiment, the present chimeric proteincomprises one or more targeting moieties directed against a T cell andone or more targeting moieties directed against a dendritic cell. In oneembodiment, the present chimeric protein comprises one or more targetingmoieties against a T cell and one or more targeting moieties directedagainst a macrophage. In one embodiment, the present chimeric proteincomprises one or more targeting moieties against a T cell and one ormore targeting moieties directed against a NK cell. For example, in anillustrative embodiment, the chimeric protein may include a targetingmoiety against CD8 and a targeting moiety against Clec9A. In anotherillustrative embodiment, the chimeric protein may include a targetingmoiety against CD8 and a targeting moiety against CD3. In anotherillustrative embodiment, the chimeric protein may include a targetingmoiety against CD8 and a targeting moiety against PD-1.

In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a B cell and one or more targetingmoieties directed against the same or another B cell. In one embodiment,the present chimeric protein comprises one or more targeting moietiesdirected against a B cell and one or more targeting moieties directedagainst a T cell. In one embodiment, the present chimeric proteincomprises one or more targeting moieties directed against a B cell andone or more targeting moieties directed against a dendritic cell. In oneembodiment, the present chimeric protein comprises one or more targetingmoieties against a B cell and one or more targeting moieties directedagainst a macrophage. In one embodiment, the present chimeric proteincomprises one or more targeting moieties against a B cell and one ormore targeting moieties directed against a NK cell.

In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a dendritic cell and one or moretargeting moieties directed against the same or another dendritic cell.In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a dendritic cell and one or moretargeting moieties directed against a T cell. In one embodiment, thepresent chimeric protein comprises one or more targeting moietiesdirected against a dendritic cell and one or more targeting moietiesdirected against a B cell. In one embodiment, the present chimericprotein comprises one or more targeting moieties against a dendriticcell and one or more targeting moieties directed against a macrophage.In one embodiment, the present chimeric protein comprises one or moretargeting moieties against a dendritic cell and one or more targetingmoieties directed against a NK cell.

In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a macrophage and one or moretargeting moieties directed against the same or another macrophage. Inone embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a macrophage and one or moretargeting moieties directed against a T cell. In one embodiment, thepresent chimeric protein comprises one or more targeting moietiesdirected against a macrophage and one or more targeting moietiesdirected against a B cell. In one embodiment, the present chimericprotein comprises one or more targeting moieties against a macrophageand one or more targeting moieties directed against a dendritic cell. Inone embodiment, the present chimeric protein comprises one or moretargeting moieties against a macrophage and one or more targetingmoieties directed against a NK cell.

In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against an NK cell and one or more targetingmoieties directed against the same or another NK cell. In oneembodiment, the present chimeric protein comprises one or more targetingmoieties directed against an NK cell and one or more targeting moietiesdirected against a T cell. In one embodiment, the present chimericprotein comprises one or more targeting moieties directed against an NKcell and one or more targeting moieties directed against a B cell. Inone embodiment, the present chimeric protein comprises one or moretargeting moieties against an NK cell and one or more targeting moietiesdirected against a macrophage. In one embodiment, the present chimericprotein comprises one or more targeting moieties against an NK cell andone or more targeting moieties directed against a dendritic cell.

In one embodiment, the present chimeric protein comprises a targetingmoiety directed against a tumor cell and a second targeting moietydirected against the same or a different tumor cell. In suchembodiments, the targeting moieties may bind to any of the tumorantigens described herein.

Targeting Moiety Formats

In various embodiments, the targeting moiety of the present chimericprotein is a protein-based agent capable of specific binding, such as anantibody or derivatives thereof. In an embodiment, the targeting moietycomprises an antibody. In various embodiments, the antibody is afull-length multimeric protein that includes two heavy chains and twolight chains. Each heavy chain includes one variable region (e.g.,V_(H)) and at least three constant regions (e.g., CH₁, CH₂ and CH₃), andeach light chain includes one variable region (V_(L)) and one constantregion (C_(L)). The variable regions determine the specificity of theantibody. Each variable region comprises three hypervariable regionsalso known as complementarity determining regions (CDRs) flanked by fourrelatively conserved framework regions (FRs). The three CDRs, referredto as CDR1, CDR2, and CDR3, contribute to the antibody bindingspecificity. In some embodiments, the antibody is a chimeric antibody.In some embodiments, the antibody is a humanized antibody.

In some embodiments, the targeting moiety comprises antibody derivativesor formats. In some embodiments, the targeting moiety of the presentchimeric protein is a single-domain antibody, a recombinantheavy-chain-only antibody (VHH), a single-chain antibody (scFv), a sharkheavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein,knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; anAnticalin; an AdNectin; an Affilin; a Microbody; a peptide aptamer; analterases; a plastic antibodies; a phylomer; a stradobodies; amaxibodies; an evibody; a fynomer, an armadillo repeat protein, a Kunitzdomain, an avimer, an atrimer, a probody, an immunobody, a triomab, atroybody; a pepbody; a vaccibody, a UniBody; affimers, a DuoBody, a Fv,a Fab, a Fab′, a F(ab′)₂, a peptide mimetic molecule, or a syntheticmolecule, as described in US Patent Nos. or Patent Publication Nos. U.S.Pat. No. 7,417,130, US 2004/132094, U.S. Pat. No. 5,831,012, US2004/023334, U.S. Pat. Nos. 7,250,297 6,818,418, US 2004/209243, U.S.Pat. Nos. 7,838,629, 7,186,524, 6,004,746, 5,475,096, US 2004/146938, US2004/157209, U.S. Pat. Nos. 6,994,982, 6,794,144, US 2010/239633, U.S.Pat. No. 7,803,907, US 2010/119446, and/or U.S. Pat. No. 7,166,697, thecontents of which are hereby incorporated by reference in theirentireties. See also, Storz MAbs. 2011 May-June; 3(3): 310-317.

In one embodiment, the targeting moiety comprises a single-domainantibody, such as VHH from, for example, an organism that produces VHHantibody such as a camelid, a shark, or a designed VHH. VHHs areantibody-derived therapeutic proteins that contain the unique structuraland functional properties of naturally-occurring heavy-chain antibodies.VHH technology is based on fully functional antibodies from camelidsthat lack light chains. These heavy-chain antibodies contain a singlevariable domain (VHH) and two constant domains (CH2 and CH3). VHHs arecommercially available under the trademark of NANOBODIES.

In various embodiments, the targeting moiety of the present chimericprotein is a protein-based agent capable of specific binding to a cellreceptor, such as a natural ligand for the cell receptor. In variousembodiments, the cell receptor is found on one or more immune cells,which can include, without limitation, T cells, cytotoxic T lymphocytes,T helper cells, natural killer (NK) cells, natural killer T (NKT) cells,anti-tumor macrophages (e.g. M1 macrophages), B cells, dendritic cells,or subsets thereof. In some embodiments, the cell receptor is found onmegakaryocytes, thrombocytes, erythrocytes, mast cells, basophils,neutrophils, eosinophils, or subsets thereof.

In some embodiments, the targeting moiety is a natural ligand such as achemokine. Exemplary chemokines that may be included in the chimericprotein of the invention include, but are not limited to, CCL1, CCL2,CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14,CCL15, CCL16, CL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24,CLL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7,CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16,CXCL17, XCL1, XCL2, CX3CL1, HCC-4, and LDGF-PBP. In an illustrativeembodiment, the targeting moiety may be XCL1 which is a chemokine thatrecognizes and binds to the dendritic cell receptor XCR1. In anotherillustrative embodiment, the targeting moiety is CCL1, which is achemokine that recognizes and binds to CCR8. In another illustrativeembodiment, the targeting moiety is CCL2, which is a chemokine thatrecognizes and binds to CCR2 or CCR9. In another illustrativeembodiment, the targeting moiety is CCL3, which is a chemokine thatrecognizes and binds to CCR1, CCR5, or CCR9. In another illustrativeembodiment, the targeting moiety is CCL4, which is a chemokine thatrecognizes and binds to CCR1 or CCR5 or CCR9. In another illustrativeembodiment, the targeting moiety is CCL5, which is a chemokine thatrecognizes and binds to CCR1 or CCR3 or CCR4 or CCR5. In anotherillustrative embodiment, the targeting moiety is CCL6, which is achemokine that recognizes and binds to CCR1. In another illustrativeembodiment, the targeting moiety is CCL7, which is a chemokine thatrecognizes and binds to CCR2 or CCR9. In another illustrativeembodiment, the targeting moiety is CCL8, which is a chemokine thatrecognizes and binds to CCR1 or CCR2 or CCR2B or CCR5 or CCR9. Inanother illustrative embodiment, the targeting moiety is CCL9, which isa chemokine that recognizes and binds to CCR1. In another illustrativeembodiment, the targeting moiety is CCL10, which is a chemokine thatrecognizes and binds to CCR1. In another illustrative embodiment, thetargeting moiety is CCL11, which is a chemokine that recognizes andbinds to CCR2 or CCR3 or CCR5 or CCR9. In another illustrativeembodiment, the targeting moiety is CCL13, which is a chemokine thatrecognizes and binds to CCR2 or CCR3 or CCR5 or CCR9. In anotherillustrative embodiment, the targeting moiety is CCL14, which is achemokine that recognizes and binds to CCR1 or CCR9. In anotherillustrative embodiment, the targeting moiety is CCL15, which is achemokine that recognizes and binds to CCR1 or CCR3. In anotherillustrative embodiment, the targeting moiety is CCL16, which is achemokine that recognizes and binds to CCR1, CCR2, CCR5, or CCR8. Inanother illustrative embodiment, the targeting moiety is CCL17, which isa chemokine that recognizes and binds to CCR4. In another illustrativeembodiment, the targeting moiety is CCL19, which is a chemokine thatrecognizes and binds to CCR7. In another illustrative embodiment, thetargeting moiety is CCL20, which is a chemokine that recognizes andbinds to CCR6. In another illustrative embodiment, the targeting moietyis CCL21, which is a chemokine that recognizes and binds to CCR7. Inanother illustrative embodiment, the targeting moiety is CCL22, which isa chemokine that recognizes and binds to CCR4. In another illustrativeembodiment, the targeting moiety is CCL23, which is a chemokine thatrecognizes and binds to CCR1. In another illustrative embodiment, thetargeting moiety is CCL24, which is a chemokine that recognizes andbinds to CCR3. In another illustrative embodiment, the targeting moietyis CCL25, which is a chemokine that recognizes and binds to CCR9. Inanother illustrative embodiment, the targeting moiety is CCL26, which isa chemokine that recognizes and binds to CCR3. In another illustrativeembodiment, the targeting moiety is CCL27, which is a chemokine thatrecognizes and binds to CCR10. In another illustrative embodiment, thetargeting moiety is CCL28, which is a chemokine that recognizes andbinds to CCR3 or CCR10. In another illustrative embodiment, thetargeting moiety is CXCL1, which is a chemokine that recognizes andbinds to CXCR1 or CXCR2. In another illustrative embodiment, thetargeting moiety is CXCL2, which is a chemokine that recognizes andbinds to CXCR2. In another illustrative embodiment, the targeting moietyis CXCL3, which is a chemokine that recognizes and binds to CXCR2. Inanother illustrative embodiment, the targeting moiety is CXCL4, which isa chemokine that recognizes and binds to CXCR3B. In another illustrativeembodiment, the targeting moiety is CXCL5, which is a chemokine thatrecognizes and binds to CXCR2. In another illustrative embodiment, thetargeting moiety is CXCL6, which is a chemokine that recognizes andbinds to CXCR1 or CXCR2. In another illustrative embodiment, thetargeting moiety is CXCL8, which is a chemokine that recognizes andbinds to CXCR1 or CXCR2. In another illustrative embodiment, thetargeting moiety is CXCL9, which is a chemokine that recognizes andbinds to CXCR3. In another illustrative embodiment, the targeting moietyis CXCL10, which is a chemokine that recognizes and binds to CXCR3. Inanother illustrative embodiment, the targeting moiety is CXCL11, whichis a chemokine that recognizes and binds to CXCR3 or CXCR7. In anotherillustrative embodiment, the targeting moiety is CXCL12, which is achemokine that recognizes and binds to CXCR4 or CXCR7. In anotherillustrative embodiment, the targeting moiety is CXCL13, which is achemokine that recognizes and binds to CXCR5. In another illustrativeembodiment, the targeting moiety is CXCL16, which is a chemokine thatrecognizes and binds to CXCR6. In another illustrative embodiment, thetargeting moiety is LDGF-PBP, which is a chemokine that recognizes andbinds to CXCR2. In another illustrative embodiment, the targeting moietyis XCL2, which is a chemokine that recognizes and binds to XCR1. Inanother illustrative embodiment, the targeting moiety is CX3CL1, whichis a chemokine that recognizes and binds to CX3CR1.

In various embodiments, the present chimeric protein comprises targetingmoieties in various combinations. In an illustrative embodiment, thepresent chimeric protein may comprise two targeting moieties, whereinboth targeting moieties are antibodies or derivatives thereof. Inanother illustrative embodiment, the present chimeric protein maycomprise two targeting moieties, wherein both targeting moieties arenatural ligands for cell receptors. In a further illustrativeembodiment, the present chimeric protein may comprise two targetingmoieties, wherein one of the targeting moieties is an antibody orderivative thereof, and the other targeting moiety is a natural ligandfor a cell receptor.

In various embodiments, the recognition domain of the present chimericprotein functionally modulates (by way of non-limitation, partially orcompletely neutralizes) the target (e.g. antigen, receptor) of interest,e.g. substantially inhibiting, reducing, or neutralizing a biologicaleffect that the antigen has. For example, various recognition domainsmay be directed against one or more tumor antigens that are activelysuppressing, or have the capacity to suppress, the immune system of, forexample, a patient bearing a tumor. For example, in some embodiments,the present chimeric protein functionally modulates immune inhibitorysignals (e.g. checkpoint inhibitors), for example, one or more of TIM-3,BTLA, PD-1, CTLA-4, B7-H4, GITR, galectin-9, HVEM, PD-L1, PD-L2, B7-H3,CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, and TMIGD2. For example, insome embodiments, the present chimeric protein is engineered to disrupt,block, reduce, and/or inhibit the transmission of an immune inhibitorysignal, by way of non-limiting example, the binding of PD-1 with PD-L1or PD-L2 and/or the binding of CTLA-4 with one or more of AP2M1, CD80,CD86, SHP-2, and PPP2R5A.

In various embodiments, the recognition domain of the present chimericprotein binds but does not functionally modulate the target (e.g.antigen, receptor) of interest, e.g. the recognition domain is, or isakin to, a binding antibody. For instance, in various embodiments, therecognition domain simply targets the antigen or receptor but does notsubstantially inhibit, reduce or functionally modulate a biologicaleffect that the antigen or receptor has. For example, some of thesmaller antibody formats described above (e.g. as compared to, forexample, full antibodies) have the ability to target hard to accessepitopes and provide a larger spectrum of specific binding locales. Invarious embodiments, the recognition domain binds an epitope that isphysically separate from an antigen or receptor site that is importantfor its biological activity (e.g. the antigen's active site).

Such non-neutralizing binding finds use in various embodiments of thepresent invention, including methods in which the present chimericprotein is used to directly or indirectly recruit active immune cells toa site of need via an effector antigen, such as any of those describedherein. For example, in various embodiments, the present chimericprotein may be used to directly or indirectly recruit cytotoxic T cellsvia CD8 to a tumor cell in a method of reducing or eliminating a tumor(e.g. the chimeric protein may comprise an anti-CD8 recognition domainand a recognition domain directed against a tumor antigen). In suchembodiments, it is desirable to directly or indirectly recruitCD8-expressing cytotoxic T cells but not to functionally modulate theCD8 activity. On the contrary, in these embodiments, CD8 signaling is animportant piece of the tumor reducing or eliminating effect. By way offurther example, in various methods of reducing or eliminating tumors,the present chimeric protein is used to directly or indirectly recruitdendritic cells (DCs) via CLEC9A (e.g. the chimeric protein may comprisean anti-CLEC9A recognition domain and a recognition domain directedagainst a tumor antigen). In such embodiments, it is desirable todirectly or indirectly recruit CLEC9A-expressing DCs but not tofunctionally modulate the CLEC9A activity. On the contrary, in theseembodiments, CLEC9A signaling is an important piece of the tumorreducing or eliminating effect.

In various embodiments, the recognition domain of the present chimericprotein binds to XCR1 e.g. on dendritic cells. For instance, therecognition domain, in some embodiments comprises all or part of XCL1 ora non-neutralizing anti-XCR1 agent.

In various embodiments, the recognition domain of the present chimericprotein binds to an immune modulatory antigen (e.g. immune stimulatoryor immune inhibitory). In various embodiments, the immune modulatoryantigen is one or more of 4-1BB, OX-40, HVEM, GITR, CD27, CD28, CD30,CD40, ICOS ligand; OX-40 ligand, LIGHT (CD258), GITR ligand, CD70, B7-1,B7-2, CD30 ligand, CD40 ligand, ICOS, ICOS ligand, CD137 ligand andTL1A. In various embodiments, such immune stimulatory antigens areexpressed on a tumor cell. In various embodiments, the recognitiondomain of the present chimeric protein binds but does not functionallymodulate such immune stimulatory antigens and therefore allowsrecruitment of cells expressing these antigens without the reduction orloss of their potential tumor reducing or eliminating capacity.

In various embodiments, the recognition domain of the present chimericprotein may be in the context of chimeric protein that comprises tworecognition domains that have neutralizing activity, or comprises tworecognition domains that have non-neutralizing (e.g. binding) activity,or comprises one recognition domain that has neutralizing activity andone recognition domain that has non-neutralizing (e.g. binding)activity.

Additional Signaling Agents

In one aspect, the present invention provides a chimeric proteincomprising one or more signaling agents (for instance, animmune-modulating agent) in addition to the modified IFN-β describedherein. In exemplary embodiments, the chimeric protein may comprise two,three, four, five, six, seven, eight, nine, ten or more signaling agentsin addition to the modified IFN-β described herein. In variousembodiments, the additional signaling agent is modified to have reducedaffinity or activity for one or more of its receptors, which allows forattenuation of activity (inclusive of agonism or antagonism) and/orprevents non-specific signaling or undesirable sequestration of thechimeric protein.

In various embodiments, the additional signaling agent is antagonisticin its wild type form and bears one or more mutations that attenuate itsantagonistic activity. In various embodiments, the additional signalingagent is antagonistic due to one or more mutations, e.g. an agonisticsignaling agent is converted to an antagonistic signaling agent and,such a converted signaling agent, optionally, also bears one or moremutations that attenuate its antagonistic activity (e.g. as described inWO 2015/007520, the entire contents of which are hereby incorporated byreference).

In various embodiments, the additional signaling agent is selected frommodified versions of cytokines, growth factors, and hormones.Illustrative examples of such cytokines, growth factors, and hormonesinclude, but are not limited to, lymphokines, monokines, traditionalpolypeptide hormones, such as human growth hormone, N-methionyl humangrowth hormone, and bovine growth hormone; parathyroid hormone;thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoproteinhormones such as follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), and luteinizing hormone (LH); hepatic growth factor;fibroblast growth factor; prolactin; placental lactogen; tumor necrosisfactor-α and tumor necrosis factor-β; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-α; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and-II; osteo inductive factors; interferons such as, for example,interferon-α, interferon-β and interferon-γ (and interferon type I, II,and III), colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF); interleukins (ILs) such as, for example, IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,and IL-18; a tumor necrosis factor such as, for example, TNF-α or TNF-β;and other polypeptide factors including, for example, LIF and kit ligand(KL). As used herein, cytokines, growth factors, and hormones includeproteins obtained from natural sources or produced from recombinantbacterial, eukaryotic or mammalian cell culture systems and biologicallyactive equivalents of the native sequence cytokines.

In some embodiments, the additional signaling agent is a modifiedversion of a growth factor selected from, but not limited to,transforming growth factors (TGFs) such as TGF-α and TGF-β, epidermalgrowth factor (EGF), insulin-like growth factor such as insulin-likegrowth factor-I and -II, fibroblast growth factor (FGF), heregulin,platelet-derived growth factor (PDGF), vascular endothelial growthfactor (VEGF).

In an embodiment, the growth factor is a modified version of afibroblast growth factor (FGF). Illustrative FGFs include, but are notlimited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10,FGF11, FGF12, FGF13, FGF14, murine FGF15, FGF16, FGF17, FGF18, FGF19,FGF20, FGF21, FGF22, and FGF23.

In an embodiment, the growth factor is a modified version of a vascularendothelial growth factor (VEGF). Illustrative VEGFs include, but arenot limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF and isoformsthereof including the various isoforms of VEGF-A such as VEGF₁₂₁,VEGF₁₂₁b, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₆₅b, VEGF₁₈₉, and VEGF₂₀₆.

In an embodiment, the growth factor is a modified version of atransforming growth factor (TGF). Illustrative TGFs include, but are notlimited to, TGF-α and TGF-β and subtypes thereof including the varioussubtypes of TGF-β including TGFβ1, TGFβ2, and TGFβ3.

In some embodiments, the additional signaling agent is a modifiedversion of a hormone selected from, but not limited to, human chorionicgonadotropin, gonadotropin releasing hormone, an androgen, an estrogen,thyroid-stimulating hormone, follicle-stimulating hormone, luteinizinghormone, prolactin, growth hormone, adrenocorticotropic hormone,antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, growthhormone releasing hormone, corticotropin-releasing hormone,somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroidhormone, glucocorticoids, mineralocorticoids, adrenaline, noradrenaline,progesterone, insulin, glucagon, amylin, calcitriol, calciferol,atrial-natriuretic peptide, gastrin, secretin, cholecystokinin,neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor (IGF),leptin, thrombopoietin, erythropoietin (EPO), and angiotensinogen.

In some embodiments, the additional signaling agent is animmune-modulating agent, e.g. one or more of an interleukin, interferon,and tumor necrosis factor.

In some embodiments, the additional signaling agent is an interleukin,including for example IL-1; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8;IL-9; IL-10; IL-11; IL-12; IL-13; IL-14; IL-15; IL-16; IL-17; IL-18;IL-19; IL-20; IL-21; IL-22; IL-23; IL-24; IL-25; IL-26; IL-27; IL-28;IL-29; IL-30; IL-31; IL-32; IL-33; IL-35; IL-36 or a fragment, variant,analogue, or family-member thereof. Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, monocytes, andmacrophages. Known functions include stimulating proliferation of immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity can be determined using assays knownin the art: Matthews et al., in Lymphokines and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp.221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20.

In some embodiments, the additional signaling agent is a modifiedversion of an interferon such as interferon types I, II, and III.Illustrative interferons, include, for example, interferon-β andinterferon-γ, interferon κ, interferon ε, interferon τ, and interferonω.

In some embodiments, the additional signaling agent is a modifiedversion of a tumor necrosis factor (TNF) or a protein in the TNF family,including but not limited to, TNF-α, TNF-β, LT-β, CD40L, CD27L, CD30L,FASL, 4-1BBL, OX40L, and TRAIL.

In various embodiments, the additional signaling agent is a modified(e.g. mutant) form of the signaling agent having one or more mutations.In various embodiments, the mutations allow for the modified signalingagent to have one or more of attenuated activity such as one or more ofreduced binding affinity, reduced endogenous activity, and reducedspecific bioactivity relative to unmodified or unmutated, i.e. the wildtype form of the signaling agent (e.g. comparing the same signalingagent in a wild type form versus a modified (e.g. mutant) form). In someembodiments, the mutations which attenuate or reduce binding or affinityinclude those mutations which substantially reduce or ablate binding oractivity. In some embodiments, the mutations which attenuate or reducebinding or affinity are different than those mutations whichsubstantially reduce or ablate binding or activity. Consequentially, invarious embodiments, the mutations allow for the signaling agent to bemore safe, e.g. have reduced systemic toxicity, reduced side effects,and reduced off-target effects relative to unmutated, i.e. wild type,signaling agent (e.g. comparing the same signaling agent in a wild typeform versus a modified (e.g. mutant) form).

In various embodiments, the additional signaling agent is modified tohave one or more mutations that reduce its binding affinity or activityfor one or more of its receptors. In some embodiments, the signalingagent is modified to have one or more mutations that substantiallyreduce or ablate binding affinity or activity for the receptors. In someembodiments, the activity provided by the wild type signaling agent isagonism at the receptor (e.g. activation of a cellular effect at a siteof therapy). For example, the wild type signaling agent may activate itsreceptor. In such embodiments, the mutations result in the modifiedsignaling agent to have reduced or ablated activating activity at thereceptor. For example, the mutations may result in the modifiedsignaling agent to deliver a reduced activating signal to a target cellor the activating signal could be ablated. In some embodiments, theactivity provided by the wild type signaling agent is antagonism at thereceptor (e.g. blocking or dampening of a cellular effect at a site oftherapy). For example, the wild type signaling agent may antagonize orinhibit the receptor. In these embodiments, the mutations result in themodified signaling agent to have a reduced or ablated antagonizingactivity at the receptor. For example, the mutations may result in themodified signaling agent to deliver a reduced inhibitory signal to atarget cell or the inhibitory signal could be ablated. In variousembodiments, the signaling agent is antagonistic due to one or moremutations, e.g. an agonistic signaling agent is converted to anantagonistic signaling agent (e.g. as described in WO 2015/007520, theentire contents of which are hereby incorporated by reference) and, sucha converted signaling agent, optionally, also bears one or moremutations that reduce its binding affinity or activity for one or moreof its receptors or that substantially reduce or ablate binding affinityor activity for one or more of its receptors.

In some embodiments, the reduced affinity or activity at the receptor isrestorable by attachment with one or more of the targeting moieties. Inother embodiments, the reduced affinity or activity at the receptor isnot substantially restorable by the activity of one or more of thetargeting moieties.

In various embodiments, the additional signaling agent is active ontarget cells because the targeting moiety(ies) compensates for themissing/insufficient binding (e.g., without limitation and/or avidity)required for substantial activation. In various embodiments, themodified signaling agent is substantially inactive en route to the siteof therapeutic activity and has its effect substantially on specificallytargeted cell types which greatly reduces undesired side effects.

In some embodiments, the additional signaling agent may include one ormore mutations that attenuate or reduce binding or affinity for onereceptor (i.e., a therapeutic receptor) and one or more mutations thatsubstantially reduce or ablate binding or activity at a second receptor.In such embodiments, these mutations may be at the same or at differentpositions (i.e., the same mutation or multiple mutations). In someembodiments, the mutation(s) that reduce binding and/or activity at onereceptor is different than the mutation(s) that substantially reduce orablate at another receptor. In some embodiments, the mutation(s) thatreduce binding and/or activity at one receptor is the same as themutation(s) that substantially reduce or ablate at another receptor. Insome embodiments, the present chimeric proteins have a modifiedsignaling agent that has both mutations that attenuate binding and/oractivity at a therapeutic receptor and therefore allow for a morecontrolled, on-target therapeutic effect (e.g. relative wild typesignaling agent) and mutations that substantially reduce or ablatebinding and/or activity at another receptor and therefore reduce sideeffects (e.g. relative to wild type signaling agent).

In some embodiments, the substantial reduction or ablation of binding oractivity is not substantially restorable with a targeting moiety. Insome embodiments, the substantial reduction or ablation of binding oractivity is restorable with a targeting moiety. In various embodiments,substantially reducing or ablating binding or activity at a secondreceptor also may prevent deleterious effects that are mediated by theother receptor. Alternatively, or in addition, substantially reducing orablating binding or activity at the other receptor causes thetherapeutic effect to improve as there is a reduced or eliminatedsequestration of the therapeutic chimeric proteins away from the site oftherapeutic action. For instance, in some embodiments, this obviates theneed of high doses of the present chimeric proteins that compensate forloss at the other receptor. Such ability to reduce dose further providesa lower likelihood of side effects.

In various embodiments, the additional modified signaling agentcomprises one or more mutations that cause the signaling agent to havereduced, substantially reduced, or ablated affinity, e.g. binding (e.g.K_(D)) and/or activation (for instance, when the modified signalingagent is an agonist of its receptor, measurable as, for example, K_(A)and/or EC₅₀) and/or inhibition (for instance, when the modifiedsignaling agent is an antagonist of its receptor, measurable as, forexample, K_(I) and/or IC₅₀), for one or more of its receptors. Invarious embodiments, the reduced affinity at the signaling agent'sreceptor allows for attenuation of activity (inclusive of agonism orantagonism). In such embodiments, the modified signaling agent has about1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or about 10%-20%, about 20%-40%, about 50%, about 40%-60%, about60%-80%, about 80%-100% of the affinity for the receptor relative to thewild type signaling agent. In some embodiments, the binding affinity isat least about 2-fold lower, about 3-fold lower, about 4-fold lower,about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-foldlower, about 9-fold lower, at least about 10-fold lower, at least about15-fold lower, at least about 20-fold lower, at least about 25-foldlower, at least about 30-fold lower, at least about 35-fold lower, atleast about 40-fold lower, at least about 45-fold lower, at least about50-fold lower, at least about 100-fold lower, at least about 150-foldlower, or about 10-50-fold lower, about 50-100-fold lower, about100-150-fold lower, about 150-200-fold lower, or more than 200-foldlower relative to the wild type signaling agent.

In embodiments wherein the chimeric protein has mutations that reducebinding at one receptor and substantially reduce or ablate binding at asecond receptor, the attenuation or reduction in binding affinity of amodified signaling agent for one receptor is less than the substantialreduction or ablation in affinity for the other receptor. In someembodiments, the attenuation or reduction in binding affinity of amodified signaling agent for one receptor is less than the substantialreduction or ablation in affinity for the other receptor by about 1%, orabout 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Invarious embodiments, substantial reduction or ablation refers to agreater reduction in binding affinity and/or activity than attenuationor reduction.

In various embodiments, the additional modified signaling agentcomprises one or more mutations that reduce the endogenous activity ofthe signaling agent to about 75%, or about 70%, or about 60%, or about50%, or about 40%, or about 30%, or about 25%, or about 20%, or about10%, or about 5%, or about 3%, or about 1%, e.g., relative to the wildtype signaling agent

In various embodiments, the additional modified signaling agentcomprises one or more mutations that cause the signaling agent to havereduced affinity and/or activity for a receptor of any one of thecytokines, growth factors, and hormones as described herein.

In some embodiments, the additional modified signaling agent comprisesone or more mutations that cause the signaling agent to have reducedaffinity for its receptor that is lower than the binding affinity of thetargeting moiety(ies) for its(their) receptor(s). In some embodiments,this binding affinity differential is between signaling agent/receptorand targeting moiety/receptor on the same cell. In some embodiments,this binding affinity differential allows for the signaling agent, e.g.mutated signaling agent, to have localized, on-target effects and tominimize off-target effects that underlie side effects that are observedwith wild type signaling agent. In some embodiments, this bindingaffinity is at least about 2-fold, or at least about 5-fold, or at leastabout 10-fold, or at least about 15-fold lower, or at least about25-fold, or at least about 50-fold lower, or at least about 100-fold, orat least about 150-fold.

Receptor binding activity may be measured using methods known in theart. For example, affinity and/or binding activity may be assessed byScatchard plot analysis and computer-fitting of binding data (e.g.Scatchard, 1949) or by reflectometric interference spectroscopy underflow through conditions, as described by Brecht et al. (1993), theentire contents of all of which are hereby incorporated by reference.

The amino acid sequences of the wild type signaling agents describedherein are well known in the art. Accordingly, in various embodimentsthe additional modified signaling agent comprises an amino acid sequencethat has at least about 60%, or at least about 61%, or at least about62%, or at least about 63%, or at least about 64%, or at least about65%, or at least about 66%, or at least about 67%, or at least about68%, or at least about 69%, or at least about 70%, or at least about71%, or at least about 72%, or at least about 73%, or at least about74%, or at least about 75%, or at least about 76%, or at least about77%, or at least about 78%, or at least about 79%, or at least about80%, or at least about 81%, or at least about 82%, or at least about83%, or at least about 84%, or at least about 85%, or at least about86%, or at least about 87%, or at least about 88%, or at least about89%, or at least about 90%, or at least about 91%, or at least about92%, or at least about 93%, or at least about 94%, or at least about95%, or at least about 96%, or at least about 97%, or at least about98%, or at least about 99% sequence identity with the known wild typeamino acid sequences of the signaling agents described herein (e.g.about 60%, or about 61%, or about 62%, or about 63%, or about 64%, orabout 65%, or about 66%, or about 67%, or about 68%, or about 69%, orabout 70%, or about 71%, or about 72%, or about 73%, or about 74%, orabout 75%, or about 76%, or about 77%, or about 78%, or about 79%, orabout 80%, or about 81%, or about 82%, or about 83%, or about 84%, orabout 85%, or about 86%, or about 87%, or about 88%, or about 89%, orabout 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%, or about 96%, or about 97%, or about 98%, or about 99%sequence identity).

In various embodiments the additional modified signaling agent comprisesan amino acid sequence that has at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withany of the sequences disclosed herein (e.g. about 60%, or about 61%, orabout 62%, or about 63%, or about 64%, or about 65%, or about 66%, orabout 67%, or about 68%, or about 69%, or about 70%, or about 71%, orabout 72%, or about 73%, or about 74%, or about 75%, or about 76%, orabout 77%, or about 78%, or about 79%, or about 80%, or about 81%, orabout 82%, or about 83%, or about 84%, or about 85%, or about 86%, orabout 87%, or about 88%, or about 89%, or about 90%, or about 91%, orabout 92%, or about 93%, or about 94%, or about 95%, or about 96%, orabout 97%, or about 98%, or about 99% sequence identity).

In various embodiments, the additional modified signaling agentcomprises an amino acid sequence having one or more amino acidmutations. In some embodiments, the one or more amino acid mutations maybe independently selected from substitutions, insertions, deletions, andtruncations.

In some embodiments, the amino acid mutations are amino acidsubstitutions, and may include conservative and/or non-conservativesubstitutions as described herein.

As described herein, the additional modified signaling agents bearmutations that affect affinity and/or activity at one or more receptors.In various embodiments, there is reduced affinity and/or activity at atherapeutic receptor, e.g. a receptor through which a desiredtherapeutic effect is mediated (e.g. agonism or antagonism). In variousembodiments, the modified signaling agents bear mutations thatsubstantially reduce or ablate affinity and/or activity at a receptor,e.g. a receptor through which a desired therapeutic effect is notmediated (e.g. as the result of promiscuity of binding). The receptorsof any modified signaling agents, e.g. one of the cytokines, growthfactors, and hormones as described herein, are known in the art.

Illustrative mutations which provide reduced affinity and/or activity(e.g. agonistic) at a receptor are found in WO 2013/107791 (e.g. withregard to interferons), WO 2015/007542 (e.g. with regard tointerleukins), and WO 2015/007903 (e.g. with regard to TNF), the entirecontents of each of which are hereby incorporated by reference.Illustrative mutations which provide reduced affinity and/or activity(e.g. antagonistic) at a therapeutic receptor are found in WO2015/007520, the entire contents of which are hereby incorporated byreference.

In some embodiments, the additional modified signaling agent comprisesone or more mutations that cause the signaling agent to have reducedaffinity and/or activity for a type I cytokine receptor, a type IIcytokine receptor, a chemokine receptor, a receptor in the TumorNecrosis Factor Receptor (TNFR) superfamily, TGF-beta Receptors, areceptor in the immunoglobulin (Ig) superfamily, and/or a receptor inthe tyrosine kinase superfamily.

In various embodiments, the receptor for the additional signaling agentis a Type I cytokine receptor. Type I cytokine receptors are known inthe art and include, but are not limited to receptors for IL2(beta-subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF,LIF, CNTF, and also the receptors for Thrombopoietin (TPO), Prolactin,and Growth hormone. Illustrative type I cytokine receptors include, butare not limited to, GM-CSF receptor, G-CSF receptor, LIF receptor, CNTFreceptor, TPO receptor, and type I IL receptors.

In various embodiments, the receptor for the additional signaling agentis a Type II cytokine receptor. Type II cytokine receptors aremultimeric receptors composed of heterologous subunits, and arereceptors mainly for interferons. This family of receptors includes, butis not limited to, receptors for interferon-α, interferon-β andinterferon-γ, IL10, IL22, and tissue factor. Illustrative type IIcytokine receptors include, but are not limited to, IFN-α receptor (e.g.IFNAR1 and IFNAR2), IFN-β receptor, IFN-γ receptor (e.g. IFNGR1 andIFNGR2), and type II IL receptors.

In various embodiments, the receptor for the additional signaling agentis a G protein-coupled receptor. Chemokine receptors are Gprotein-coupled receptors with seven transmembrane structure and coupledto G-protein for signal transduction. Chemokine receptors include, butare not limited to, CC chemokine receptors, CXC chemokine receptors,CX3C chemokine receptors, and XC chemokine receptor (XCR1). Exemplarychemokine receptors include, but are not limited to, CCR1, CCR2, CCR3,CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B,CXCR4, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR1.

In various embodiments, the receptor for the additional signaling agentis a TNFR family member. Tumor necrosis factor receptor (TNFR) familymembers share a cysteine-rich domain (CRD) formed of three disulfidebonds surrounding a core motif of CXXCXXC creating an elongatedmolecule. Exemplary tumor necrosis factor receptor family membersinclude: CDI 20a (TNFRSFIA), CD 120b (TNFRSFIB), Lymphotoxin betareceptor (LTBR, TNFRSF3), CD 134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS(FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30(TNFRSF8), CD137 (TNFRSF9), TNFRSFIOA (TNFRSFIOA), TNFRSFIOB,(TNFRSFIOB), TNFRSFIOC (TNFRSFIOC), TNFRSFIOD (TNFRSFIOD), RANK(TNFRSFIIA), Osteoprotegerin (TNFRSFIIB), TNFRSF12A (TNFRSF12A),TNFRSF13B (TNFRSF13B), TNFRSF13C (TNFRSF13C), TNFRSF14 (TNFRSF14), Nervegrowth factor receptor (NGFR, TNFRSF16), TNFRSF17 (TNFRSF17), TNFRSF18(TNFRSF18), TNFRSF19 (TNFRSF19), TNFRSF21 (TNFRSF21), and TNFRSF25(TNFRSF25).

In various embodiments, the receptor for the additional signaling agentis a TGF-beta receptor. TGF-beta receptors are single passserine/threonine kinase receptors. TGF-beta receptors include, but arenot limited to, TGFBR1, TGFBR2, and TGFBR3.

In various embodiments, the receptor for the additional signaling agentis an Ig superfamily receptor. Receptors in the immunoglobulin (Ig)superfamily share structural homology with immunoglobulins. Receptors inthe Ig superfamily include, but are not limited to, interleukin-1receptors, CSF-1R, PDGFR (e.g. PDGFRA and PDGFRB), and SCFR.

In various embodiments, the receptor for the additional signaling agentis a tyrosine kinase superfamily receptor. Receptors in the tyrosinekinase superfamily are well known in the art. There are about 58 knownreceptor tyrosine kinases (RTKs), grouped into 20 subfamilies. Receptorsin the tyrosine kinase superfamily include, but are not limited to, FGFreceptors and their various isoforms such as FGFR1, FGFR2, FGFR3, FGFR4,and FGFR5.

In an embodiment, the additional modified signaling agent is anotherinterferon β. In such embodiments, the modified interferon β agent alsohas reduced affinity and/or activity for the IFN-α/β receptor (IFNAR),i.e., IFNAR1 and/or IFNAR2 chains. In some embodiments, the modifiedinterferon β agent has substantially reduced or ablated affinity and/oractivity for the IFN-α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2chains.

In an embodiment, the additional modified signaling agent is interferonγ. In such embodiments, the modified interferon γ agent has reducedaffinity and/or activity for the interferon-gamma receptor (IFNGR),i.e., IFNGR1 and IFNGR2 chains. In some embodiments, the modifiedinterferon γ agent has substantially reduced or ablated affinity and/oractivity for the interferon-gamma receptor (IFNGR), i.e., IFNGR1 and/orIFNGR2 chains.

In some embodiments, the additional modified signaling agent is vascularendothelial growth factor (VEGF). VEGF is a potent growth factor thatplays major roles in physiological but also pathological angiogenesis,regulates vascular permeability and can act as a growth factor on cellsexpressing VEGF receptors. Additional functions include, among others,stimulation of cell migration in macrophage lineage and endothelialcells. Several members of the VEGF family of growth factors exist, aswell as at least three receptors (VEGFR-1, VEGFR-2, and VEGFR-3).Members of the VEGF family can bind and activate more than one VEGFRtype. For example, VEGF-A binds VEGFR-1 and -2, while VEGF-C can bindVEGFR-2 and -3. VEGFR-1 and -2 activation regulates angiogenesis whileVEGFR-3 activation is associated with lymphangiogenesis. The majorpro-angiogenic signal is generated from activation of VEGFR-2. VEGFR-1activation has been reported to be possibly associated with negativerole in angiogenesis. It has also been reported that VEGFR-1 signalingis important for progression of tumors in vivo via bone marrow-derivedVEGFR-1 positive cells (contributing to formation of premetastatic nichein the bone). Several therapies based on VEGF-A directed/neutralizingtherapeutic antibodies have been developed, primarily for use intreatment of various human tumors relying on angiogenesis. These are notwithout side effects though. This may not be surprising considering thatthese operate as general, non-cell/tissue specific VEGF/VEGFRinteraction inhibitors. Hence, it would be desirable to restrict VEGF(e.g. VEGF-A)/VEGFR-2 inhibition to specific target cells (e.g. tumorvasculature endothelial cells).

In some embodiments, the VEGF is VEGF-A, VEGF-B, VEFG-C, VEGF-D, orVEGF-E and isoforms thereof including the various isoforms of VEGF-Asuch as VEGF₁₂₁, VEGF₁₂₁b, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₆₅b, VEGF₁₈₉, andVEGF₂₀₆. In some embodiments, the modified signaling agent has reducedaffinity and/or activity for VEGFR-1 (Flt-1) and/or VEGFR-2 (KDR/Flk-1).In some embodiments, the modified signaling agent has substantiallyreduced or ablated affinity and/or activity for VEGFR-1 (Flt-1) and/orVEGFR-2 (KDR/Flk-1). In an embodiment, the modified signaling agent hasreduced affinity and/or activity for VEGFR-2 (KDR/Flk-1) and/orsubstantially reduced or ablated affinity and/or activity for VEGFR-1(Flt-1). Such an embodiment finds use, for example, in wound healingmethods or treatment of ischmia-related diseases (without wishing to bebound by theory, mediated by VEGFR-2's effects on endothelial cellfunction and angiogenesis). In various embodiments, binding to VEGFR-1(Flt-1), which is linked to cancers and pro-inflammatory activities, isavoided. In various embodiments, VEGFR-1 (Flt-1) acts a decoy receptorand therefore substantially reduces or ablates affinity at this receptoravoids sequestration of the therapeutic agent. In an embodiment, themodified signaling agent has substantially reduced or ablated affinityand/or activity for VEGFR-1 (Flt-1) and/or substantially reduced orablated affinity and/or activity for VEGFR-2 (KDR/Flk-1). In someembodiments, the VEGF is VEGF-C or VEGF-D. In such embodiments, themodified signaling agent has reduced affinity and/or activity forVEGFR-3. Alternatively, the modified signaling agent has substantiallyreduced or ablated affinity and/or activity for VEGFR-3.

Proangiogenic therapies are also important in various diseases (e.g.ischemic heart disease, bleeding etc.), and include VEGF-basedtherapeutics. Activation of VEGFR-2 is proangiogenic (acting onendothelial cells). Activation of VEFGR-1 can cause stimulation ofmigration of inflammatory cells (including, for example, macrophages)and lead to inflammation associated hypervascular permeability.Activation of VEFGR-1 can also promote bone marrow associated tumorniche formation. Thus, VEGF based therapeutic selective for VEGFR-2activation would be desirable in this case. In addition, cell specifictargeting, e.g. to endothelial cells, would be desirable.

In some embodiments, the additional modified signaling agent has reducedaffinity and/or activity (e.g. antagonistic) for VEGFR-2 and/or hassubstantially reduced or ablated affinity and/or activity for VEGFR-1.When targeted to tumor vasculature endothelial cells via a targetingmoiety that binds to a tumor endothelial cell marker (e.g. PSMA andothers), such construct inhibits VEGFR-2 activation specifically on suchmarker-positive cells, while not activating VEGFR-1 en route and ontarget cells (if activity ablated), thus eliminating induction ofinflammatory responses, for example. This would provide a more selectiveand safe anti-angiogenic therapy for many tumor types as compared toVEGF-A neutralizing therapies.

In some embodiments, the additional modified signaling agent has reducedaffinity and/or activity (e.g. agonistic) for VEGFR-2 and/or hassubstantially reduced or ablated affinity and/or activity for VEGFR-1.Through targeting to vascular endothelial cells, such construct, in someembodiments, promotes angiogenesis without causing VEGFR-1 associatedinduction of inflammatory responses. Hence, such a construct would havetargeted proangiogenic effects with substantially reduced risk of sideeffects caused by systemic activation of VEGFR-2 as well as VEGR-1.

In an illustrative embodiment, the modified signaling agent is VEGF₁₆₅,which has the amino acid sequence:

VEGF 165 (wild type) (SEQ ID NO: 180)APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQ LELNERTCRCDKPRR

In another illustrative embodiment, the additional modified signalingagent is VEGF_(165b), which has the amino acid sequence:

VEGF 165b (wild type) (SEQ ID NO: 181)APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQ LELNERTCRSLTRKD

In these embodiments, the modified signaling agent has a mutation atamino acid I83 (e.g., a substitution mutation at I83, e.g., I83K, I83R,or I83H). Without wishing to be bound by theory, it is believed thatsuch mutations may result in reduced receptor binding affinity. See, forexample, U.S. Pat. No. 9,078,860, the entire contents of which arehereby incorporated by reference.

In an embodiment, the additional modified signaling agent is TNF-α. TNFis a pleiotropic cytokine with many diverse functions, includingregulation of cell growth, differentiation, apoptosis, tumorigenesis,viral replication, autoimmunity, immune cell functions and trafficking,inflammation, and septic shock. It binds to two distinct membranereceptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits avery broad expression pattern whereas TNFR2 is expressed preferentiallyon certain populations of lymphocytes, Tregs, endothelial cells, certainneurons, microglia, cardiac myocytes and mesenchymal stem cells. Verydistinct biological pathways are activated in response to receptoractivation, although there is also some overlap. As a general rule,without wishing to be bound by theory, TNFR1 signaling is associatedwith induction of apoptosis (cell death) and TNFR2 signaling isassociated with activation of cell survival signals (e.g. activation ofNFkB pathway). Administration of TNF is systemically toxic, and this islargely due to TNFR1 engagement. However, it should be noted thatactivation of TNFR2 is also associated with a broad range of activitiesand, as with TNFR1, in the context of developing TNF based therapeutics,control over TNF targeting and activity is important.

In some embodiments, the additional modified signaling agent has reducedaffinity and/or activity for TNFR1 and/or TNFR2. In some embodiments,the modified signaling agent has substantially reduced or ablatedaffinity and/or activity for TNFR1 and/or TNFR2. TNFR1 is expressed inmost tissues, and is involved in cell death signaling while, bycontrast, TNFR2 is involved in cell survival signaling. Accordingly, inembodiments directed to methods of treating cancer, the modifiedsignaling agent has reduced affinity and/or activity for TNFR1 and/orsubstantially reduced or ablated affinity and/or activity for TNFR2. Inthese embodiments, the chimeric proteins may be targeted to a cell forwhich apoptosis is desired, e.g. a tumor cell or a tumor vasculatureendothelial cell. In embodiments directed to methods of promoting cellsurvival, for example, in neurogenesis for the treatment ofneurodegenerative disorders, the modified signaling agent has reducedaffinity and/or activity for TNFR2 and/or substantially reduced orablated affinity and/or activity for TNFR1. Stated another way, thepresent chimeric proteins, in some embodiments, comprise modified TNF-αagent that allows of favoring either death or survival signals.

In some embodiments, the chimeric protein has a modified TNF havingreduced affinity and/or activity for TNFR1 and/or substantially reducedor ablated affinity and/or activity for TNFR2. Such a chimera, in someembodiments, is a more potent inducer of apoptosis as compared to a wildtype TNF and/or a chimera bearing only mutation(s) causing reducedaffinity and/or activity for TNFR1. Such a chimera, in some embodiments,finds use in inducing tumor cell death or a tumor vasculatureendothelial cell death (e.g. in the treatment of cancers). Also, in someembodiments, these chimeras avoid or reduce activation of T_(reg) cellsvia TNFR2, for example, thus further supporting TNFR1-mediated antitumoractivity in vivo.

In some embodiments, the chimeric protein has a modified TNF havingreduced affinity and/or activity for TNFR2 and/or substantially reducedor ablated affinity and/or activity for TNFR1. Such a chimera, in someembodiments, is a more potent activator of cell survival in some celltypes, which may be a specific therapeutic objective in various diseasesettings, including without limitation, stimulation of neurogenesis. Inaddition, such a TNFR2-favoring chimeras also are useful in thetreatment of autoimmune diseases (e.g. Crohn's, diabetes, MS, colitisetc. and many others described herein). In some embodiments, the chimerais targeted to auto-reactive T cells. In some embodiments, the chimerapromotes T_(reg) cell activation and indirect suppression of cytotoxic Tcells.

In some embodiments, the chimera causes the death of auto-reactive Tcells, e.g. by activation of TNFR2 and/or avoidance of TNFR1 (e.g. amodified TNF having reduced affinity and/or activity for TNFR2 and/orsubstantially reduced or ablated affinity and/or activity for TNFR1).Without wishing to be bound by theory these auto-reactive T cells, havetheir apoptosis/survival signals altered e.g. by NFkB pathwayactivity/signaling alterations.

In some embodiments, a TNFR2 based chimera has additional therapeuticapplications in diseases, including various autoimmune diseases, heartdisease, de-myelinating and neurodegenerative disorders, and infectiousdisease, among others.

In an embodiment, the wild type TNF-α has the amino acid sequence of:

TNF-α (SEQ ID NO: 182)VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQV YFGIIAL

In such embodiments, the modified TNF-α agent has mutations at one ormore amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146and 147 which produces a modified TNF-α with reduced receptor bindingaffinity. See, for example, U.S. Pat. No. 7,993,636, the entire contentsof which are hereby incorporated by reference.

In some embodiments, the modified human TNF-α moiety has mutations atone or more amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87,V91, I97, T105, P106, A109, P113, Y115, E127, N137, D143, and A145, asdescribed, for example, in WO/2015/007903, the entire contents of whichis hereby incorporated by reference (numbering according to the humanTNF sequence, Genbank accession number BAG70306, version BAG70306.1 GI:197692685). In some embodiments, the modified human TNF-α moiety hassubstitution mutations selected from R32G, N34G, Q67G, H73G, L75G, L75A,L75S, T77A, S86G, Y87Q, Y87L, Y87A, Y87F, V91G, V91A, I97A, I97Q, I97S,T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G andA145T. In an embodiment, the human TNF-α moiety has a mutation selectedfrom Y87Q, Y87L, Y87A, and Y87F. In another embodiment, the human TNF-αmoiety has a mutation selected from I97A, I97Q, and I97S. In a furtherembodiment, the human TNF-α moiety has a mutation selected from Y115Aand Y115G.

In some embodiments, the modified TNF-α agent has one or more mutationsselected from N39Y, S147Y, and Y87H, as described in WO2008/124086, theentire contents of which is hereby incorporated by reference.

In an embodiment, the additional modified signaling agent is TNF-β.TNF-β can form a homotrimer or a heterotrimer with LT-β (LT-α1β2). Insome embodiments, the modified signaling agent has substantially reducedor ablated affinity and/or activity for TNFR1 and/or TNFR2 and/or herpesvirus entry mediator (HEVM) and/or LT-βR.

In an embodiment, the wild type TNF-β has the amino acid sequence of:

TNF-beta (SEQ ID NO: 183)LPGVGLTPSAAQTARQHPKMHLAHSNLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTH TDGIPHLVLSPSTVFFGAFAL

In such embodiments, the modified TNF-β agent may comprise mutations atone or more amino acids at positions 106-113, which produce a modifiedTNF-β with reduced receptor binding affinity to TNFR2. In an embodiment,the modified signaling agent has one or more substitution mutations atamino acid positions 106-113. In illustrative embodiments, thesubstitution mutations are selected from Q107E, Q107D, S106E, S106D,Q107R, Q107N, Q107E/S106E, Q107E/S106D, Q107D/S106E, and Q107D/S106D. Inanother embodiment, the modified signaling agent has an insertion ofabout 1 to about 3 amino acids at positions 106-113.

In some embodiments, the additional modified agent is a TNF familymember (e.g. TNF-alpha, TNF-beta) which can be a single chain trimericversion as described in WO 2015/007903, the entire contents of which areincorporated by reference.

In some embodiments, the modified agent is a TNF family member (e.g.TNF-alpha, TNF-beta) which has reduced affinity and/or activity, i.e.antagonistic activity (e.g. natural antagonistic activity orantagonistic activity that is the result of one or more mutations, see,e.g., WO 2015/007520, the entire contents of which are herebyincorporated by reference) at TNFR1. In these embodiments, the modifiedagent is a TNF family member (e.g. TNF-alpha, TNF-beta) which also,optionally, has substantially reduced or ablated affinity and/oractivity for TNFR2. In some embodiments, the modified agent is a TNFfamily member (e.g. TNF-alpha, TNF-beta) which has reduced affinityand/or activity, i.e. antagonistic activity (e.g. natural antagonisticactivity or antagonistic activity that is the result of one or moremutations, see, e.g., WO 2015/007520, the entire contents of which arehereby incorporated by reference) at TNFR2. In these embodiments, themodified agent is a TNF family member (e.g. TNF-alpha, TNF-beta) whichalso, optionally, has substantially reduced or ablated affinity and/oractivity for TNFR1. The constructs of such embodiments find use in, forexample, methods of dampening TNF response in a cell specific manner. Insome embodiments, the antagonistic TNF family member (e.g. TNF-alpha,TNF-beta) is a single chain trimeric version as described in WO2015/007903.

In an embodiment, the additional modified signaling agent is TRAIL. Insome embodiments, the modified TRAIL agent has reduced affinity and/oractivity for DR4 (TRAIL-RI) and/or DR5 (TRAIL-RII) and/or DcR1 and/orDcR2. In some embodiments, the modified TRAIL agent has substantiallyreduced or ablated affinity and/or activity for DR4 (TRAIL-RI) and/orDR5 (TRAIL-RII) and/or DcR1 and/or DcR2.

In an embodiment, the wild type TRAIL has the amino acid sequence of:

TRAIL (SEQ ID NO: 184)MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG

In such embodiments, the modified TRAIL agent may comprise a mutation atamino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209,T214-1220,K224-A226, W231, E236-L239, E249-K251, T261-H264 and H270-E271(Numbering based on the human sequence, Genbank accession numberNP_003801, version 10 NP_003801.1, GI: 4507593; see above).

In an embodiment, the additional modified signaling agent is TGFα. Insuch embodiments, the modified TGFα agent has reduced affinity and/oractivity for the epidermal growth factor receptor (EGFR). In someembodiments, the modified TGFα agent has substantially reduced orablated affinity and/or activity for the epidermal growth factorreceptor (EGFR).

In an embodiment, the additional modified signaling agent is TGFβ. Insuch embodiments, the modified signaling agent has reduced affinityand/or activity for TGFBR1 and/or TGFBR2. In some embodiments, themodified signaling agent has substantially reduced or ablated affinityand/or activity for TGFBR1 and/or TGFBR2. In some embodiments, themodified signaling agent optionally has reduced or substantially reducedor ablated affinity and/or activity for TGFBR3 which, without wishing tobe bound by theory, may act as a reservoir of ligand for TGF-betareceptors. In some embodiments, the TGFβ may favor TGFBR1 over TGFBR2 orTGFBR2 over TGFBR1. Similarly, LAP, without wishing to be bound bytheory, may act as a reservoir of ligand for TGF-beta receptors. In someembodiments, the modified signaling agent has reduced affinity and/oractivity for TGFBR1 and/or TGFBR2 and/or substantially reduced orablated affinity and/or activity for Latency Associated Peptide (LAP).In some embodiments, such chimeras find use in Camurati-Engelmanndisease, or other diseases associated with inappropriate TGFβ signaling.

In some embodiments, the additional modified agent is a TGF familymember (e.g. TGFα, TGFβ) which has reduced affinity and/or activity,i.e. antagonistic activity (e.g. natural antagonistic activity orantagonistic activity that is the result of one or more mutations, see,e.g., WO 2015/007520, the entire contents of which are herebyincorporated by reference) at one or more of TGFBR1, TGFBR2, TGFBR3. Inthese embodiments, the modified agent is a TGF family member (e.g. TGFα,TGFβ) which also, optionally, has substantially reduced or ablatedaffinity and/or activity at one or more of TGFBR1, TGFBR2, TGFBR3.

In some embodiments, the additional modified agent is a TGF familymember (e.g. TGFα, TGFβ) which has reduced affinity and/or activity,i.e. antagonistic activity (e.g. natural antagonistic activity orantagonistic activity that is the result of one or more mutations, see,e.g., WO 2015/007520, the entire contents of which are herebyincorporated by reference) at TGFBR1 and/or TGFBR2. In theseembodiments, the modified agent is a TGF family member (e.g. TGFα, TGFβ)which also, optionally, has substantially reduced or ablated affinityand/or activity at TGFBR3.

In an embodiment, the additional modified signaling agent is IL-1. In anembodiment, the modified signaling agent is IL-1α or IL-1β. In someembodiments, the modified signaling agent has reduced affinity and/oractivity for IL-1R1 and/or IL-1RAcP. In some embodiments, the modifiedsignaling agent has substantially reduced or ablated affinity and/oractivity for IL-1R1 and/or IL-1RAcP. In some embodiments, the modifiedsignaling agent has reduced affinity and/or activity for IL-1R2. In someembodiments, the modified signaling agent has substantially reduced orablated affinity and/or activity for IL-1R2. For instance, in someembodiments, the present modified IL-1 agents avoid interaction atIL-1R2 and therefore substantially reduce its function as a decoy and/orsink for therapeutic agents.

In an embodiment, the wild type IL-1β has the amino acid sequence of:

IL-1 beta (mature form, wild type) (SEQ ID NO: 185)APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQF VSS

IL1 is a proinflammatory cytokine and an important immune systemregulator. It is a potent activator of CD4 T cell responses, increasesproportion of Th17 cells and expansion of IFNγ and IL-4 producing cells.IL-1 is also a potent regulator of CD8⁺ T cells, enhancingantigen-specific CD8⁺ T cell expansion, differentiation, migration toperiphery and memory. IL-1 receptors comprise IL-1R1 and IL-1R2. Bindingto and signaling through the IL-1R1 constitutes the mechanism wherebyIL-1 mediates many of its biological (and pathological) activities.IL1-R2 can function as a decoy receptor, thereby reducing IL-1availability for interaction and signaling through the IL-1R1.

In some embodiments, the modified IL-1 has reduced affinity and/oractivity (e.g. agonistic activity) for IL-1R1. In some embodiments, themodified IL-1 has substantially reduced or ablated affinity and/oractivity for IL-1R2. In such embodiments, there is restorableIL-1/IL-1R1 signaling and prevention of loss of therapeutic chimeras atIL-R2 and therefore a reduction in dose of IL-1 that is required (e.g.relative to wild type or a chimera bearing only an attenuation mutationfor IL-R1). Such constructs find use in, for example, methods oftreating cancer, including, for example, stimulating the immune systemto mount an anti-cancer response.

In some embodiments, the modified IL-1 has reduced affinity and/oractivity (e.g. antagonistic activity, e.g. natural antagonistic activityor antagonistic activity that is the result of one or more mutations,see, e.g., WO 2015/007520, the entire contents of which are herebyincorporated by reference) for IL-1R1. In some embodiments, the modifiedIL-1 has substantially reduced or ablated affinity and/or activity forIL-1R2. In such embodiments, there is the IL-1/IL-1R1 signaling is notrestorable and prevention of loss of therapeutic chimeras at IL-R2 andtherefore a reduction in dose of IL-1 that is required (e.g. relative towild type or a chimera bearing only an attenuation mutation for IL-R1).Such constructs find use in, for example, methods of treating autoimmunediseases, including, for example, suppressing the immune system.

In such embodiments, the modified signaling agent has a deletion ofamino acids 52-54 which produces a modified human IL-1β with reducedbinding affinity for type I IL-1R and reduced biological activity. See,for example, WO 1994/000491, the entire contents of which are herebyincorporated by reference. In some embodiments, the modified human IL-1βhas one or more substitution mutations selected from A117G/P118G, R120X,L122A, T125G/L126G, R127G, Q130X, Q131G, K132A, S137G/Q138Y, L145G,H146X, L145A/L147A, Q148X, Q148G/Q150G, Q150G/D151A, M152G, F162A,F162A/Q164E, F166A, Q164E/E167K, N169G/D170G, I172A, V174A, K208E,K209X, K209A/K210A, K219X, E221X, E221 S/N224A, N224S/K225S, E244K,N245Q (where X can be any change in amino acid, e.g., a non-conservativechange), which exhibit reduced binding to IL-1R, as described, forexample, in WO2015/007542 and WO/2015/007536, the entire contents ofwhich is hereby incorporated by reference (numbering base on the humanIL-1 β sequence, Genbank accession number NP_000567, versionNP-000567.1, GI: 10835145). In some embodiments, the modified humanIL-1β may have one or more mutations selected from R120A, R120G, Q130A,Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A,K209D, K219S, K219Q, E221S and E221K. In an embodiment, the modifiedhuman IL-1β comprises the mutations Q131G and Q148G. In an embodiment,the modified human IL-1β comprises the mutations Q148G and K208E. In anembodiment, the modified human IL-1β comprises the mutations R120G andQ131G. In an embodiment, the modified human IL-1β comprises themutations R120G and H146G. In an embodiment, the modified human IL-1βcomprises the mutations R120G and K208E. In an embodiment, the modifiedhuman IL-1β comprises the mutations R120G, F162A, and Q164E.

In an embodiment, the additional modified signaling agent is IL-2. Insuch an embodiment, the modified signaling agent has reduced affinityand/or activity for IL-2Rα and/or IL-2Rβ and/or IL-2Rγ. In someembodiments, the modified signaling agent has reduced affinity and/oractivity for IL-2Rβ and/or IL-2Rγ. In some embodiments, the modifiedsignaling agent has substantially reduced or ablated affinity and/oractivity for IL-2Rα. Such embodiments may be relevant for treatment ofcancer, for instance when the modified IL-2 is agonistic at IL-2Rβand/or IL-2Rγ. For instance, the present constructs may favor attenuatedactivation of CD8⁺ T cells (which can provide an anti-tumor effect),which have IL2 receptors β and γ and disfavor T_(regs) (which canprovide an immune suppressive, pro-tumor effect), which have IL2receptors α, β, and γ. Further, in some embodiments, the preferences forIL-2Rβ and/or IL-2Rγ over IL-2Rα avoid IL-2 side effects such aspulmonary edema. Also, IL-2-based chimeras are useful for the treatmentof autoimmune diseases, for instance when the modified IL-2 isantagonistic (e.g. natural antagonistic activity or antagonisticactivity that is the result of one or more mutations, see, e.g., WO2015/007520, the entire contents of which are hereby incorporated byreference) at IL-2Rβ and/or IL-2Rγ. For instance, the present constructsmay favor attenuated suppression of CD8⁺ T cells (and therefore dampenthe immune response), which have IL2 receptors β and γ and disfavorT_(regs) which have IL2 receptors α, β, and γ. Alternatively, in someembodiments, the chimeras bearing IL-2 favor the activation of T_(regs),and therefore immune suppression, and activation of disfavor of CD8⁺ Tcells. For instance, these constructs find use in the treatment ofdiseases or diseases that would benefit from immune suppression, e.g.autoimmune disorders.

In some embodiments, the chimeric protein has targeting moieties asdescribed herein directed to CD8⁺ T cells as well as a modified IL-2agent having reduced affinity and/or activity for IL-2Rβ and/or IL-2Rγand/or substantially reduced or ablated affinity and/or activity forIL-2Rα. In some embodiments, these constructs provide targeted CD8⁺ Tcell activity and are generally inactive (or have substantially reducedactivity) towards T_(reg) cells. In some embodiments, such constructshave enhanced immune stimulatory effect compared to wild type IL-2(e.g., without wishing to be bound by theory, by not stimulating Tregs),whilst eliminating or reducing the systemic toxicity associated withIL-2.

In an embodiment, the wild type IL-2 has the amino acid sequence of:

IL-2 (mature form, wild type) (SEQ ID NO: 186)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

In such embodiments, the modified IL-2 agent has one or more mutationsat amino acids L72 (L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D,L72R, or L72K), F42 (F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D,F42R, or F42K) and Y45 (Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D,Y45R or Y45K). Without wishing to be bound by theory, it is believedthat these modified IL-2 agents have reduced affinity for thehigh-affinity IL-2 receptor and preserves affinity to theintermediate-affinity IL-2 receptor, as compared to the wild-type IL-2.See, for example, US Patent Publication No. 2012/0244112, the entirecontents of which are hereby incorporated by reference.

In an embodiment, the additional modified signaling agent is IL-3. Insome embodiments, the modified signaling agent has reduced affinityand/or activity for the IL-3 receptor, which is a heterodimer with aunique alpha chain paired with the common beta (beta c or CD131)subunit. In some embodiments, the modified signaling agent hassubstantially reduced or ablated affinity and/or activity for the IL-3receptor, which is a heterodimer with a unique alpha chain paired withthe common beta (beta c or CD131) subunit.

In an embodiment, the additional modified signaling agent is IL-4. Insuch an embodiment, the modified signaling agent has reduced affinityand/or activity for type 1 and/or type 2 IL-4 receptors. In such anembodiment, the modified signaling agent has substantially reduced orablated affinity and/or activity for type 1 and/or type 2 IL-4receptors. Type 1 IL-4 receptors are composed of the IL-4Rα subunit witha common γ chain and specifically bind IL-4. Type 2 IL-4 receptorsinclude an IL-4Rα subunit bound to a different subunit known asIL-13Rα1. In some embodiments, the modified signaling agent hassubstantially reduced or ablated affinity and/or activity the type 2IL-4 receptors.

In an embodiment, the wild type IL-4 has the amino acid sequence of:

IL-4 (mature form, wild type) (SEQ ID NO: 187)HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS

In such embodiments, the modified IL-4 agent has one or more mutationsat amino acids R121 (R121A, R121D, R121E, R121F, R121H, R121I, R121K,R121N, R121P, R121T, R121W), E122 (E122F), Y124 (Y124A, Y124Q, Y124R,Y124S, Y124T) and S125 (S125A). Without wishing to be bound by theory,it is believed that these modified IL-4 agents maintain the activitymediated by the type I receptor, but significantly reduces thebiological activity mediated by the other receptors. See, for example,U.S. Pat. No. 6,433,157, the entire contents of which are herebyincorporated by reference.

In an embodiment, the additional modified signaling agent is IL-6. IL-6signals through a cell-surface type I cytokine receptor complexincluding the ligand-binding IL-6R chain (CD126), and thesignal-transducing component gp130. IL-6 may also bind to a soluble formof IL-6R (sIL-6R), which is the extracellular portion of IL-6R. ThesIL-6R/IL-6 complex may be involved in neurites outgrowth and survivalof neurons and, hence, may be important in nerve regeneration throughremyelination. Accordingly, in some embodiments, the modified signalingagent has reduced affinity and/or activity for IL-6R/gp130 and/orsIL-6R. In some embodiments, the modified signaling agent hassubstantially reduced or ablated affinity and/or activity forIL-6R/gp130 and/or sIL-6R.

In an embodiment, the wild type IL-6 has the amino acid sequence of:

IL-6 (mature form, wild type) (SEQ ID NO: 188)APVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLTTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM

In such embodiments, the modified signaling agent has one or moremutations at amino acids 58, 160, 163, 171 or 177. Without wishing to bebound by theory, it is believed that these modified IL-6 agents exhibitreduced binding affinity to IL-6Ralpha and reduced biological activity.See, for example, WO 97/10338, the entire contents of which are herebyincorporated by reference.

In an embodiment, the additional modified signaling agent is IL-10. Insuch an embodiment, the modified signaling agent has reduced affinityand/or activity for IL-10 receptor-1 and IL-10 receptor-2. In someembodiments, the modified signaling agent has substantially reduced orablated affinity and/or activity for IL-10 receptor-1 and IL-10receptor-2

In an embodiment, the additional modified signaling agent is IL-11. Insuch an embodiment, the modified signaling agent has reduced affinityand/or activity for IL-11Rα and/or IL-11Rβ and/or gp130. In such anembodiment, the modified signaling agent has substantially reduced orablated affinity and/or activity for IL-11Rα and/or IL-11Rβ and/orgp130.

In an embodiment, the additional modified signaling agent is IL-12. Insuch an embodiment, the modified signaling agent has reduced affinityand/or activity for IL-12Rβ1 and/or IL-12Rβ2. In such an embodiment, themodified signaling agent has substantially reduced or ablated affinityand/or activity for IL-12Rβ1 and/or IL-12Rβ2.

In an embodiment, the additional modified signaling agent is IL-13. Insuch an embodiment, the modified signaling agent has reduced affinityand/or activity for the IL-4 receptor (IL-4Rα) and IL-13Rα1. In someembodiments, the modified signaling agent has substantially reduced orablated affinity and/or activity for IL-4 receptor (IL-4Rα) or IL-13Rα1.

In an embodiment, the wild type IL-13 has the amino acid sequence of:

IL-13 (mature form, wild type) (SEQ ID NO: 189)SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDL LLHLKKLFREGRFN

In such embodiments, the modified IL-13 agent has one or more mutationsat amino acids 13, 16, 17, 66, 69, 99, 102, 104, 105, 106, 107, 108,109, 112, 113 and 114. Without wishing to be bound by theory, it isbelieved that these modified IL-13 agents exhibit reduced biologicalactivity. See, for example, WO 2002/018422, the entire contents of whichare hereby incorporated by reference.

In an embodiment, the additional modified signaling agent is IL-18. Insome embodiments, the modified signaling agent has reduced affinityand/or activity for IL-18Rα and/or IL-18Rβ. In some embodiments, themodified signaling agent has substantially reduced or ablated affinityand/or activity for IL-18Rα and/or IL-18Rβ. In some embodiments, themodified signaling agent has substantially reduced or ablated affinityand/or activity for IL-18Rα type II, which is an isoform of IL-18Rα thatlacks the TIR domain required for signaling.

In an embodiment, the wild type IL-18 has the amino acid sequence of:

IL-18 (wild type) (SEQ ID NO: 190)MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDL

In such embodiments, the modified IL-18 agent may comprise one or moremutations in amino acids or amino acid regions selected from Y37-K44,R49-Q54, D59-R63, E67-C74, R80, M87-A97, N 127-K129, Q139-M149,K165-K171, R183 and Q190-N191, as described in WO/2015/007542, theentire contents of which are hereby incorporated by reference (numberingbased on the human IL-18 sequence, Genbank accession number AAV38697,version AAV38697.1, GI: 54696650).

In an embodiment, the additional modified signaling agent is IL-33. Insuch an embodiment, the modified signaling agent has reduced affinityand/or activity for the ST-2 receptor and IL-1RAcP. In some embodiments,the modified signaling agent has substantially reduced or ablatedaffinity and/or activity for the ST-2 receptor and IL-1RAcP.

In an embodiment, the wild type IL-33 has the amino acid sequence of:

(SEQ ID NO: 191) MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALI KVDSSENLCTENILFKLSET

In such embodiments, the modified IL-33 agent may comprise one or moremutations in amino acids or amino acid regions selected from I113-Y122,S127-E139, E144-D157, Y163-M183, E200, Q215, L220-C227 and T260-E269, asdescribed in WO/2015/007542, the entire contents of which are herebyincorporated by reference (numbering based on the human sequence,Genbank accession number NP_254274, version NP_254274.1, GI:15559209).

In an embodiment, the modified signaling agent is epidermal growthfactor (EGF). EGF is a member of a family of potent growth factors.Members include EGF, HB-EGF, and others such as TGFalpha, amphiregulin,neuregulins, epiregulin, betacellulin. EGF family receptors include EGFR(ErbB1), ErbB2, ErbB3 and ErbB4. These may function as homodimericand/or heterodimeric receptor subtypes. The different EGF family membersexhibit differential selectivity for the various receptor subtypes. Forexample, EGF associates with ErbB1/ErbB1, ErbB1/ErbB2, ErbB4/ErbB2 andsome other heterodimeric subtypes. HB-EGF has a similar pattern,although it also associates with ErbB4/4. Modulation of EGF (EGF-like)growth factor signaling, positively or negatively, is of considerabletherapeutic interest. For example, inhibition of EGFRs signaling is ofinterest in the treatment of various cancers where EGFR signalingconstitutes a major growth promoting signal. Alternatively, stimulationof EGFRs signaling is of therapeutic interest in, for example, promotingwound healing (acute and chronic), oral mucositis (a major side-effectof various cancer therapies, including, without limitation radiationtherapy).

In some embodiments, the additional modified signaling agent has reducedaffinity and/or activity for ErbB1, ErbB2, ErbB3, and/or ErbB4. Suchembodiments find use, for example, in methods of treating wounds. Insome embodiments, the modified signaling agent binds to one or moreErbB1, ErbB2, ErbB3, and ErbB4 and antagonizes the activity of thereceptor. In such embodiments, the modified signaling agent has reducedaffinity and/or activity for ErbB1, ErbB2, ErbB3, and/or ErbB4 whichallows for the activity of the receptor to be antagonized in anattenuated fashion. Such embodiments find use in, for example,treatments of cancer. In an embodiment, the modified signaling agent hasreduced affinity and/or activity for ErbB1. ErbB1 is the therapeutictarget of kinase inhibitors—most have side effects because they are notvery selective (e.g., gefitinib, erlotinib, afatinib, brigatinib andicotinib). In some embodiments, attenuated antagonistic ErbB1 signalingis more on-target and has less side effects than other agents targetingreceptors for EGF.

In some embodiments, the additional modified signaling agent has reducedaffinity and/or activity (e.g. antagonistic e.g. natural antagonisticactivity or antagonistic activity that is the result of one or moremutations, see, e.g., WO 2015/007520, the entire contents of which arehereby incorporated by reference) for ErbB1 and/or substantially reducedor ablated affinity and/or activity for ErbB4 or other subtypes it mayinteract with. Through specific targeting via the targeting moiety,cell-selective suppression (antagonism e.g. natural antagonisticactivity or antagonistic activity that is the result of one or moremutations, see, e.g., WO 2015/007520, the entire contents of which arehereby incorporated by reference) of ErbB1/ErbB1 receptor activationwould be achieved—while not engaging other receptor subtypes potentiallyassociated with inhibition-associated side effects. Hence, in contrastto EGFR kinase inhibitors, which inhibit EGFR activity in all cell typesin the body, such a construct would provide a cell-selective (e.g.,tumor cell with activated EGFR signaling due to amplification ofreceptor, overexpression etc.) anti-EGFR (ErbB1) drug effect withreduced side effects.

In some embodiments, the additional modified signaling agent has reducedaffinity and/or activity (e.g. agonistic) for ErbB4 and/or othersubtypes it may interact with. Through targeting to specific targetcells through the targeting moiety, a selective activation of ErbB1signaling is achieved (e.g. epithelial cells). Such a construct findsuse, in some embodiments, in the treatment of wounds (promoting wouldhealing) with reduced side effects, especially for treatment of chronicconditions and application other than topical application of atherapeutic (e.g. systemic wound healing).

In an embodiment, the modified signaling agent is insulin or insulinanalogs. In some embodiments, the modified insulin or insulin analog hasreduced affinity and/or activity for the insulin receptor and/or IGF1 orIGF2 receptor. In some embodiments, the modified insulin or insulinanalog has substantially reduced or ablated affinity and/or activity forthe insulin receptor and/or IGF1 or IGF2 receptor. Attenuated responseat the insulin receptor allows for the control of diabetes, obesity,metabolic disorders and the like while directing away from IGF1 or IGF2receptor avoids pro-cancer effects.

In an embodiment, the modified signaling agent is insulin-like growthfactor-I or insulin-like growth factor-II (IGF-1 or IGF-2). In anembodiment, the modified signaling agent is IGF-1. In such anembodiment, the modified signaling agent has reduced affinity and/oractivity for the insulin receptor and/or IGF1 receptor. In anembodiment, the modified signaling agent may bind to the IGF1 receptorand antagonize the activity of the receptor. In such an embodiment, themodified signaling agent has reduced affinity and/or activity for IGF1receptor which allows for the activity of the receptor to be antagonizedin an attenuated fashion. In some embodiments, the modified signalingagent has substantially reduced or ablated affinity and/or activity forthe insulin receptor and/or IGF1 receptor. In some embodiments, themodified signaling agent has reduced affinity and/or activity for IGF2receptor which allows for the activity of the receptor to be antagonizedin an attenuated fashion. In an embodiment, the modified signaling agenthas substantially reduced or ablated affinity and/or activity for theinsulin receptor and accordingly does not interfere with insulinsignaling. In various embodiments, this applies to cancer treatment. Invarious embodiments, the present agents may prevent IR isoform A fromcausing resistance to cancer treatments.

In an embodiment, the modified signaling agent is EPO. In variousembodiments, the modified EPO agent has reduced affinity and/or activityfor the EPO receptor (EPOR) receptor and/or the ephrin receptor (EphR)relative to wild type EPO or other EPO based agents described herein. Insome embodiments, the modified EPO agent has substantially reduced orablated affinity and/or activity for the EPO receptor (EPOR) receptorand/or the Eph receptor (EphR). Illustrative EPO receptors include, butare not limited to, an EPOR homodimer or an EPOR/CD131 heterodimer. Alsoincluded as an EPO receptor is beta-common receptor (βcR). IllustrativeEph receptors include, but are not limited to, EPHA1, EPHA2, EPHA3,EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3,EPHB4, EPHB5, and EPHB6. In some embodiments, the modified EPO proteincomprises one or more mutations that cause the EPO protein to havereduced affinity for receptors that comprise one or more different EPOreceptors or Eph receptors (e.g. heterodimer, heterotrimers, etc.,including by way of non-limitation: EPOR-EPHB4, EPOR-βcR-EPOR). Alsoprovided are the receptors of EP Patent Publication No. 2492355 theentire contents of which are hereby incorporated by reference, includingby way of non-limitation, NEPORs.

In an embodiment, the human EPO has the amino acid sequence of (thesignal peptide is underlined):

(SEQ ID NO: 192) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

In an embodiment, the human EPO protein is the mature form of EPO (withthe signal peptide being cleaved off) which is a glycoprotein of 166amino acid residues having the sequence of:

(SEQ ID NO: 193) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLR GKLKLYTGEACRTGDR

The structure of the human EPO protein is predicted to comprisefour-helix bundles including helices A, B, C, and D. In variousembodiments, the modified EPO protein comprises one or more mutationslocated in four regions of the EPO protein which are important forbioactivity, i.e., amino acid residues 10-20, 44-51, 96-108, and142-156. In some embodiments, the one or more mutations are located atresidues 11-15, 44-51, 100-108, and 147-151. These residues arelocalized to helix A (Val11, Arg14, and Tyr15), helix C (Ser100, Arg103,Ser104, and Leu108), helix D (Asn147, Arg150, Gly151, and Leu155), andthe A/B connecting loop (residues 42-51). In some embodiments, themodified EPO protein comprises mutations in residues between amino acids41-52 and amino acids 147, 150, 151, and 155. Without wishing to bebound by theory, it is believed that mutations of these residues havesubstantial effects on both receptor binding and in vitro biologicalactivity. In some embodiments, the modified EPO protein comprisesmutations at residues 11, 14, 15, 100, 103, 104, and 108. Withoutwishing to be bound by theory, it is believed that mutations of theseresidues have modest effects on receptor binding activity and muchgreater effects on in vitro biological activity. Illustrativesubstitutions include, but are not limited to, one or more of Val11Ser,Arg14Ala, Arg14Gln, Tyr15IIe, Pro42Asn, Thr44IIe, Lys45Asp, Val46Ala,Tyr51Phe, Ser100Glu, Ser100Thr, Arg103Ala, Ser104IIe, Ser104Ala,Leu108Lys, Asn147Lys, Arg150Ala, Gly151Ala, and Leu155Ala.

In some embodiments, the modified EPO protein comprises mutations thateffect bioactivity and not binding, e.g. those listed in Eliot, et al.Mapping of the Active Site of Recombinant Human Erythropoietin Jan. 15,1997; Blood: 89 (2), the entire contents of which are herebyincorporated by reference.

In some embodiments, the modified EPO protein comprises one or moremutations involving surface residues of the EPO protein which areinvolved in receptor contact. Without wishing to be bound by theory, itis believed that mutations of these surface residues are less likely toaffect protein folding thereby retaining some biological activity.Illustrative surface residues that may be mutated include, but are notlimited to, residues 147 and 150. In illustrative embodiments, themutations are substitutions including, one or more of N147A, N147K,R150A and R150E.

In some embodiments, the modified EPO protein comprises one or moremutations at residues N59, E62, L67, and L70, and one or more mutationsthat affect disulfide bond formation. Without wishing to be bound bytheory, it is believed that these mutations affect folding and/or arepredicted be in buried positions and thus affects biological activityindirectly.

In an embodiment, the modified EPO protein comprises a K20E substitutionwhich significantly reduces receptor binding. See Elliott, et al.,(1997) Blood, 89:493-502, the entire contents of which are herebyincorporated by reference.

Additional EPO mutations that may be incorporated into the chimeric EPOprotein of the invention are disclosed in, for example, Elliott, et al.,(1997) Blood, 89:493-502, the entire contents of which are herebyincorporated by reference and Taylor et al., (2010) PEDS, 23(4):251-260, the entire contents of which are hereby incorporated byreference.

In various embodiments, the signaling agent is a toxin or toxic enzyme.In some embodiments, the toxin or toxic enzyme is derived from plantsand bacteria. Illustrative toxins or toxic enzymes include, but are notlimited to, the diphtheria toxin, Pseudomonas toxin, anthrax toxin,ribosome-inactivating proteins (RIPs) such as ricin and saporin,modeccin, abrin, gelonin, and poke weed antiviral protein. Additionaltoxins include those disclosed in Matthew et al., (2009) Cancer Sci100(8): 1359-65, the entire disclosures are hereby incorporated byreference. In such embodiments, the chimeric proteins of the inventionmay be utilized to induce cell death in cell-type specific manner. Insuch embodiments, the toxin may be modified, e.g. mutated, to reduceaffinity and/or activity of the toxin for an attenuated effect, asdescribed with other signaling agents herein.

Linkers

In some embodiments, the present chimeric protein optionally comprisesone or more linkers. In some embodiments, the present chimeric proteincomprises a linker connecting the targeting moiety and the signalingagent (e.g., modified IFN-β). In some embodiments, the present chimericprotein comprises a linker within the signaling agent (e.g., modifiedIFN-β).

In some embodiments vectors encoding the present chimeric proteinslinked as a single nucleotide sequence to any of the linkers describedherein are provided and may be used to prepare such chimeric proteins.

In some embodiments, the linker length allows for efficient binding of atargeting moiety and the signaling agent (e.g., modified IFN-β) to theirreceptors. For instance, in some embodiments, the linker length allowsfor efficient binding of one of the targeting moieties and the signalingagent to receptors on the same cell.

In some embodiments the linker length is at least equal to the minimumdistance between the binding sites of one of the targeting moieties andthe signaling agent to receptors on the same cell. In some embodimentsthe linker length is at least twice, or three times, or four times, orfive times, or ten times, or twenty times, or 25 times, or 50 times, orone hundred times, or more the minimum distance between the bindingsites of one of the targeting moieties and the signaling agent toreceptors on the same cell.

As described herein, the linker length allows for efficient binding ofone of the targeting moieties and the signaling agent to receptors onthe same cell, the binding being sequential, e.g. targetingmoiety/receptor binding preceding signaling agent/receptor binding.

In some embodiments, there are two linkers in a single chimera, eachconnecting the signaling agent to a targeting moiety. In variousembodiments, the linkers have lengths that allow for the formation of asite that has a disease cell and an effector cell without sterichindrance that would prevent modulation of the either cell.

The invention contemplates the use of a variety of linker sequences. Invarious embodiments, the linker may be derived from naturally-occurringmulti-domain proteins or are empirical linkers as described, forexample, in Chichili et al., (2013), Protein Sci. 22(2):153-167, Chen etal., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contentsof which are hereby incorporated by reference. In some embodiments, thelinker may be designed using linker designing databases and computerprograms such as those described in Chen et al., (2013), Adv Drug DelivRev. 65(10):1357-1369 and Crasto et al., (2000), Protein Eng.13(5):309-312, the entire contents of which are hereby incorporated byreference. In various embodiments, the linker may be functional. Forexample, without limitation, the linker may function to improve thefolding and/or stability, improve the expression, improve thepharmacokinetics, and/or improve the bioactivity of the present chimericprotein.

In some embodiments, the linker is a polypeptide. In some embodiments,the linker is less than about 100 amino acids long. For example, thelinker may be less than about 100, about 95, about 90, about 85, about80, about 75, about 70, about 65, about 60, about 55, about 50, about45, about 40, about 35, about 30, about 25, about 20, about 19, about18, about 17, about 16, about 15, about 14, about 13, about 12, about11, about 10, about 9, about 8, about 7, about 6, about 5, about 4,about 3, or about 2 amino acids long. In some embodiments, the linker isa polypeptide. In some embodiments, the linker is greater than about 100amino acids long. For example, the linker may be greater than about 100,about 95, about 90, about 85, about 80, about 75, about 70, about 65,about 60, about 55, about 50, about 45, about 40, about 35, about 30,about 25, about 20, about 19, about 18, about 17, about 16, about 15,about 14, about 13, about 12, about 11, about 10, about 9, about 8,about 7, about 6, about 5, about 4, about 3, or about 2 amino acidslong. In some embodiments, the linker is flexible. In anotherembodiment, the linker is rigid.

In some embodiments directed to chimeric proteins having two or moretargeting moieties, a linker connects the two targeting moieties to eachother and this linker has a short length and a linker connects atargeting moiety and a signaling agent this linker is longer than thelinker connecting the two targeting moieties. For example, thedifference in amino acid length between the linker connecting the twotargeting moieties and the linker connecting a targeting moiety and asignaling agent may be about 100, about 95, about 90, about 85, about80, about 75, about 70, about 65, about 60, about 55, about 50, about45, about 40, about 35, about 30, about 25, about 20, about 19, about18, about 17, about 16, about 15, about 14, about 13, about 12, about11, about 10, about 9, about 8, about 7, about 6, about 5, about 4,about 3, or about 2 amino acids.

In various embodiments, the linker is substantially comprised of glycineand serine residues (e.g. about 30%, or about 40%, or about 50%, orabout 60%, or about 70%, or about 80%, or about 90%, or about 95%, orabout 97% glycines and serines). For example, in some embodiments, thelinker is (Gly₄Ser)_(n), where n is from about 1 to about 8, e.g. 1, 2,3, 4, 5, 6, 7, or 8. In an embodiment, the linker sequence isGGSGGSGGGGSGGGGS (SEQ ID NO: 194). Additional illustrative linkersinclude, but are not limited to, linkers having the sequence LE, GGGGS(SEQ ID NO: 195), (GGGGS)_(n)(n=1-4) (SEQ ID NO: 196), (Gly)₈(SEQ ID NO:197), (Gly)₆(SEQ ID NO: 198), (EAAAK)_(n)(n=1-3) (SEQ ID NO: 199),A(EAAAK)_(n)A (n=2-5) (SEQ ID NO: 200), AEAAAKEAAAKA (SEQ ID NO: 201),A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 202), PAPAP (SEQ ID NO: 203),KESGSVSSEQLAQFRSLD (SEQ ID NO: 204), EGKSSGSGSESKST (SEQ ID NO: 205),GSAGSAAGSGEF (SEQ ID NO: 206), and (XP)_(n), with X designating anyamino acid, e.g., Ala, Lys, or Glu. In various embodiments, the linkeris GGS.

In some embodiments, the linker is one or more of GGGSE (SEQ ID NO:207), GSESG (SEQ ID NO: 208), GSEGS (SEQ ID NO: 209),GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 210), and a linker ofrandomly placed G, S, and E every 4 amino acid intervals.

In some embodiments, the linker is a hinge region of an antibody (e.g.,of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2,IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linkeris a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE,inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4, and IgA1 andIgA2)). The hinge region, found in IgG, IgA, IgD, and IgE classantibodies, acts as a flexible spacer, allowing the Fab portion to movefreely in space. In contrast to the constant regions, the hinge domainsare structurally diverse, varying in both sequence and length amongimmunoglobulin classes and subclasses. For example, the length andflexibility of the hinge region varies among the IgG subclasses. Thehinge region of IgG1 encompasses amino acids 216-231 and, because it isfreely flexible, the Fab fragments can rotate about their axes ofsymmetry and move within a sphere centered at the first of twointer-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1,with 12 amino acid residues and four disulfide bridges. The hinge regionof IgG2 lacks a glycine residue, is relatively short, and contains arigid poly-proline double helix, stabilized by extra inter-heavy chaindisulfide bridges. These properties restrict the flexibility of the IgG2molecule. IgG3 differs from the other subclasses by its unique extendedhinge region (about four times as long as the IgG1 hinge), containing 62amino acids (including 21 prolines and 11 cysteines), forming aninflexible poly-proline double helix. In IgG3, the Fab fragments arerelatively far away from the Fc fragment, giving the molecule a greaterflexibility. The elongated hinge in IgG3 is also responsible for itshigher molecular weight compared to the other subclasses. The hingeregion of IgG4 is shorter than that of IgG1 and its flexibility isintermediate between that of IgG1 and IgG2. The flexibility of the hingeregions reportedly decreases in the order IgG3>IgG1>IgG4>IgG2.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. See Shin etal., 1992 Immunological Reviews 130:87. The upper hinge region includesamino acids from the carboxyl end of C_(H1) to the first residue in thehinge that restricts motion, generally the first cysteine residue thatforms an interchain disulfide bond between the two heavy chains. Thelength of the upper hinge region correlates with the segmentalflexibility of the antibody. The core hinge region contains theinter-heavy chain disulfide bridges, and the lower hinge region joinsthe amino terminal end of the C_(H2) domain and includes residues inC_(H2). Id. The core hinge region of wild-type human IgG1 contains thesequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bondformation, results in a cyclic octapeptide believed to act as a pivot,thus conferring flexibility. In various embodiments, the present linkercomprises, one, or two, or three of the upper hinge region, the coreregion, and the lower hinge region of any antibody (e.g., of IgG, IgA,IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4,and IgA1 and IgA2)). The hinge region may also contain one or moreglycosylation sites, which include a number of structurally distincttypes of sites for carbohydrate attachment. For example, IgA1 containsfive glycosylation sites within a 17-amino-acid segment of the hingeregion, conferring resistance of the hinge region polypeptide tointestinal proteases, considered an advantageous property for asecretory immunoglobulin. In various embodiments, the linker of thepresent invention comprises one or more glycosylation sites. In variousembodiments, the linker is a hinge-CH2-CH3 domain of a human IgG4antibody.

If desired, the present chimeric protein can be linked to an antibody Fcregion, comprising one or both of C_(H)2 and C_(H)3 domains, andoptionally a hinge region. For example, vectors encoding the presentchimeric proteins linked as a single nucleotide sequence to an Fc regioncan be used to prepare such polypeptides.

In some embodiments, the linker is a synthetic linker such as PEG.

In various embodiments, the linker may be functional. For example,without limitation, the linker may function to improve the foldingand/or stability, improve the expression, improve the pharmacokinetics,and/or improve the bioactivity of the present chimeric protein. Inanother example, the linker may function to target the chimeric proteinto a particular cell type or location.

In various embodiments, each of the chimeric proteins may by conjugatedand/or fused with another agent to extend half-life or otherwise improvepharmacodynamic and pharmacokinetic properties. In some embodiments, thechimeric proteins may be fused or conjugated with one or more of PEG,XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., humanserum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP,transferrin, and the like. In some embodiments, the chimeric protein maybe fused or conjugated with an antibody or an antibody fragment such asan Fc fragment. For example, the chimeric protein may be fused to eitherthe N-terminus or the C-terminus of the Fc domain of humanimmunoglobulin (Ig) G. In various embodiments, each of the individualchimeric proteins is fused to one or more of the agents described inBioDrugs (2015) 29:215-239, the entire contents of which are herebyincorporated by reference.

Production of Chimeric Proteins

Methods for producing the chimeric proteins of the invention aredescribed herein. For example, DNA sequences encoding the chimericproteins of the invention (e.g., DNA sequences encoding the modifiedsignaling agent (e.g., modified IFN-β) and the targeting moiety and thelinker) can be chemically synthesized using methods known in the art.Synthetic DNA sequences can be ligated to other appropriate nucleotidesequences, including, e.g., expression control sequences, to producegene expression constructs encoding the desired chimeric proteins.Accordingly, in various embodiments, the present invention provides forisolated nucleic acids comprising a nucleotide sequence encoding thechimeric protein of the invention.

Nucleic acids encoding the chimeric protein of the invention can beincorporated (ligated) into expression vectors, which can be introducedinto host cells through transfection, transformation, or transductiontechniques. For example, nucleic acids encoding the chimeric protein ofthe invention can be introduced into host cells by retroviraltransduction. Illustrative host cells are E. coli cells, Chinese hamsterovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells.Transformed host cells can be grown under conditions that permit thehost cells to express the genes that encode the chimeric protein of theinvention. Accordingly, in various embodiments, the present inventionprovides expression vectors comprising nucleic acids that encode thechimeric protein of the invention. In various embodiments, the presentinvention additional provides host cells comprising such expressionvectors.

Specific expression and purification conditions will vary depending uponthe expression system employed. For example, if a gene is to beexpressed in E. coli, it is first cloned into an expression vector bypositioning the engineered gene downstream from a suitable bacterialpromoter, e.g., Trp or Tac, and a prokaryotic signal sequence. Inanother example, if the engineered gene is to be expressed in eukaryotichost cells, e.g., CHO cells, it is first inserted into an expressionvector containing for example, a suitable eukaryotic promoter, asecretion signal, enhancers, and various introns. The gene construct canbe introduced into the host cells using transfection, transformation, ortransduction techniques.

The chimeric protein of the invention can be produced by growing a hostcell transfected with an expression vector encoding the chimeric proteinunder conditions that permit expression of the protein. Followingexpression, the protein can be harvested and purified using techniqueswell known in the art, e.g., affinity tags such asglutathione-S-transferase (GST) and histidine tags or by chromatography.

Accordingly, in various embodiments, the present invention provides fora nucleic acid encoding a chimeric protein of the present invention. Invarious embodiments, the present invention provides for a host cellcomprising a nucleic acid encoding a chimeric protein of the presentinvention.

Pharmaceutically Acceptable Salts and Excipients

The chimeric proteins described herein can possess a sufficiently basicfunctional group, which can react with an inorganic or organic acid, ora carboxyl group, which can react with an inorganic or organic base, toform a pharmaceutically acceptable salt. A pharmaceutically acceptableacid addition salt is formed from a pharmaceutically acceptable acid, asis well known in the art. Such salts include the pharmaceuticallyacceptable salts listed in, for example, Journal of PharmaceuticalScience, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts;Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.),Verlag, Zurich (Switzerland) 2002, which are hereby incorporated byreference in their entirety.

Pharmaceutically acceptable salts include, by way of non-limitingexample, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate,chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate,phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate,hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate,heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate,mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate,phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate,chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate,methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,naphthalene-1,5-sulfonate, xylenesulfonate, and tartarate salts.

The term “pharmaceutically acceptable salt” also refers to a salt of thecompositions of the present invention having an acidic functional group,such as a carboxylic acid functional group, and a base. Suitable basesinclude, but are not limited to, hydroxides of alkali metals such assodium, potassium, and lithium; hydroxides of alkaline earth metal suchas calcium and magnesium; hydroxides of other metals, such as aluminumand zinc; ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine;tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such asmono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such asarginine, lysine, and the like.

In some embodiments, the compositions described herein are in the formof a pharmaceutically acceptable salt.

Pharmaceutical Compositions and Formulations

In various embodiments, the present invention pertains to pharmaceuticalcompositions comprising the chimeric proteins described herein and apharmaceutically acceptable carrier or excipient. Any pharmaceuticalcompositions described herein can be administered to a subject as acomponent of a composition that comprises a pharmaceutically acceptablecarrier or vehicle. Such compositions can optionally comprise a suitableamount of a pharmaceutically acceptable excipient so as to provide theform for proper administration.

In various embodiments, pharmaceutical excipients can be liquids, suchas water and oils, including those of petroleum, animal, vegetable, orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. The pharmaceutical excipients can be, for example,saline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, urea and the like. In addition, auxiliary, stabilizing,thickening, lubricating, and coloring agents can be used. In oneembodiment, the pharmaceutically acceptable excipients are sterile whenadministered to a subject. Water is a useful excipient when any agentdescribed herein is administered intravenously. Saline solutions andaqueous dextrose and glycerol solutions can also be employed as liquidexcipients, specifically for injectable solutions. Suitablepharmaceutical excipients also include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Any agent describedherein, if desired, can also comprise minor amounts of wetting oremulsifying agents, or pH buffering agents. Other examples of suitablepharmaceutical excipients are described in Remington's PharmaceuticalSciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995),incorporated herein by reference.

The present invention includes the described pharmaceutical compositions(and/or additional therapeutic agents) in various formulations. Anyinventive pharmaceutical composition (and/or additional therapeuticagents) described herein can take the form of solutions, suspensions,emulsion, drops, tablets, pills, pellets, capsules, capsules containingliquids, gelatin capsules, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, lyophilizedpowder, frozen suspension, dessicated powder, or any other form suitablefor use. In one embodiment, the composition is in the form of a capsule.In another embodiment, the composition is in the form of a tablet. Inyet another embodiment, the pharmaceutical composition is formulated inthe form of a soft-gel capsule. In a further embodiment, thepharmaceutical composition is formulated in the form of a gelatincapsule. In yet another embodiment, the pharmaceutical composition isformulated as a liquid.

Where necessary, the inventive pharmaceutical compositions (and/oradditional agents) can also include a solubilizing agent. Also, theagents can be delivered with a suitable vehicle or delivery device asknown in the art. Combination therapies outlined herein can beco-delivered in a single delivery vehicle or delivery device.

The formulations comprising the inventive pharmaceutical compositions(and/or additional agents) of the present invention may conveniently bepresented in unit dosage forms and may be prepared by any of the methodswell known in the art of pharmacy. Such methods generally include thestep of bringing the therapeutic agents into association with a carrier,which constitutes one or more accessory ingredients. Typically, theformulations are prepared by uniformly and intimately bringing thetherapeutic agent into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct into dosage forms of the desired formulation (e.g., wet or drygranulation, powder blends, etc., followed by tableting usingconventional methods known in the art).

In various embodiments, any pharmaceutical compositions (and/oradditional agents) described herein is formulated in accordance withroutine procedures as a composition adapted for a mode of administrationdescribed herein.

Routes of administration include, for example: oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, sublingual, intranasal, intracerebral, intravaginal,transdermal, rectally, by inhalation, or topically. Administration canbe local or systemic. In some embodiments, the administering is effectedorally. In another embodiment, the administration is by parenteralinjection. The mode of administration can be left to the discretion ofthe practitioner, and depends in-part upon the site of the medicalcondition. In most instances, administration results in the release ofany agent described herein into the bloodstream.

In one embodiment, the chimeric protein described herein is formulatedin accordance with routine procedures as a composition adapted for oraladministration. Compositions for oral delivery can be in the form oftablets, lozenges, aqueous or oily suspensions, granules, powders,emulsions, capsules, syrups, or elixirs, for example. Orallyadministered compositions can comprise one or more agents, for example,sweetening agents such as fructose, aspartame or saccharin; flavoringagents such as peppermint, oil of wintergreen, or cherry; coloringagents; and preserving agents, to provide a pharmaceutically palatablepreparation. Moreover, where in tablet or pill form, the compositionscan be coated to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over anextended period of time. Selectively permeable membranes surrounding anosmotically active driving any chimeric proteins described herein arealso suitable for orally administered compositions. In these latterplatforms, fluid from the environment surrounding the capsule is imbibedby the driving compound, which swells to displace the agent or agentcomposition through an aperture. These delivery platforms can provide anessentially zero order delivery profile as opposed to the spikedprofiles of immediate release formulations. A time-delay material suchas glycerol monostearate or glycerol stearate can also be useful. Oralcompositions can include standard excipients such as mannitol, lactose,starch, magnesium stearate, sodium saccharin, cellulose, and magnesiumcarbonate. In one embodiment, the excipients are of pharmaceuticalgrade. Suspensions, in addition to the active compounds, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth,etc., and mixtures thereof.

Dosage forms suitable for parenteral administration (e.g. intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g. lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents known in the art. Formulation components suitable forparenteral administration include a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as EDTA; buffers such asacetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The carrier should be stable under theconditions of manufacture and storage, and should be preserved againstmicroorganisms. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol), and suitablemixtures thereof.

The compositions provided herein, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e.,“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Any inventive pharmaceutical compositions (and/or additional agents)described herein can be administered by controlled-release orsustained-release means or by delivery devices that are well known tothose of ordinary skill in the art. Examples include, but are notlimited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767;5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of whichis incorporated herein by reference in its entirety. Such dosage formscan be useful for providing controlled- or sustained-release of one ormore active ingredients using, for example, hydropropyl cellulose,hydropropylmethyl cellulose, polyvinylpyrrolidone, other polymermatrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, liposomes, microspheres, or a combinationthereof to provide the desired release profile in varying proportions.Suitable controlled- or sustained-release formulations known to thoseskilled in the art, including those described herein, can be readilyselected for use with the active ingredients of the agents describedherein. The invention thus provides single unit dosage forms suitablefor oral administration such as, but not limited to, tablets, capsules,gelcaps, and caplets that are adapted for controlled- orsustained-release.

Controlled- or sustained-release of an active ingredient can bestimulated by various conditions, including but not limited to, changesin pH, changes in temperature, stimulation by an appropriate wavelengthof light, concentration or availability of enzymes, concentration oravailability of water, or other physiological conditions or compounds.

In another embodiment, a controlled-release system can be placed inproximity of the target area to be treated, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled-release systems discussed in the review by Langer,1990, Science 249:1527-1533) may be used.

Pharmaceutical formulations preferably are sterile. Sterilization can beaccomplished, for example, by filtration through sterile filtrationmembranes. Where the composition is lyophilized, filter sterilizationcan be conducted prior to or following lyophilization andreconstitution.

Administration and Dosage

It will be appreciated that the actual dose of the chimeric protein tobe administered according to the present invention will vary accordingto the particular dosage form, and the mode of administration. Manyfactors that may modify the action of the chimeric protein (e.g., bodyweight, gender, diet, time of administration, route of administration,rate of excretion, condition of the subject, drug combinations, geneticdisposition and reaction sensitivities) can be taken into account bythose skilled in the art. Administration can be carried out continuouslyor in one or more discrete doses within the maximum tolerated dose.Optimal administration rates for a given set of conditions can beascertained by those skilled in the art using conventional dosageadministration tests.

In some embodiments, a suitable dosage of the chimeric protein is in arange of about 0.01 mg/kg to about 10 g/kg of body weight of thesubject, about 0.01 mg/kg to about 1 g/kg of body weight of the subject,about 0.01 mg/kg to about 100 mg/kg of body weight of the subject, about0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example,about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg,about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg,about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg,about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg,about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg,about 8 mg/kg, about 9 mg/kg, about 10 mg/kg body weight, about 100mg/kg body weight, about 1 g/kg of body weight, about 10 g/kg of bodyweight, inclusive of all values and ranges therebetween.

Individual doses of the chimeric protein can be administered in unitdosage forms (e.g., tablets or capsules) containing, for example, fromabout 0.01 mg to about 100 g, from about 0.01 mg to about 75 g, fromabout 0.01 mg to about 50 g, from about 0.01 mg to about 25 g, about0.01 mg to about 10 g, about 0.01 mg to about 7.5 g, about 0.01 mg toabout 5 g, about 0.01 mg to about 2.5 g, about 0.01 mg to about 1 g,about 0.01 mg to about 100 mg, from about 0.1 mg to about 100 mg, fromabout 0.1 mg to about 90 mg, from about 0.1 mg to about 80 mg, fromabout 0.1 mg to about 70 mg, from about 0.1 mg to about 60 mg, fromabout 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg activeingredient, from about 0.1 mg to about 30 mg, from about 0.1 mg to about20 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg to about 5mg, from about 0.1 mg to about 3 mg, from about 0.1 mg to about 1 mg perunit dosage form, or from about 5 mg to about 80 mg per unit dosageform. For example, a unit dosage form can be about 0.01 mg, about 0.02mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg,about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg about 10 mg,about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg,about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about95 mg, about 100 mg, about 200 mg, about 500 mg, about 1 g, about 2.5 g,about 5 g, about 10 g, about 25 g, about 50 g, about 75 g, about 100 g,inclusive of all values and ranges therebetween.

In one embodiment, the chimeric protein is administered at an amount offrom about 0.01 mg to about 100 g daily, from about 0.01 mg to about 75g daily, from about 0.01 mg to about 50 g daily, from about 0.01 mg toabout 25 g daily, from about 0.01 mg to about 10 g daily, from about0.01 mg to about 7.5 g daily, from about 0.01 mg to about 5 g daily,from about 0.01 mg to about 2.5 g daily, from about 0.01 mg to about 1 gdaily, from about 0.01 mg to about 100 mg daily, from about 0.1 mg toabout 100 mg daily, from about 0.1 mg to about 95 mg daily, from about0.1 mg to about 90 mg daily, from about 0.1 mg to about 85 mg daily,from about 0.1 mg to about 80 mg daily, from about 0.1 mg to about 75 mgdaily, from about 0.1 mg to about 70 mg daily, from about 0.1 mg toabout 65 mg daily, from about 0.1 mg to about 60 mg daily, from about0.1 mg to about 55 mg daily, from about 0.1 mg to about 50 mg daily,from about 0.1 mg to about 45 mg daily, from about 0.1 mg to about 40 mgdaily, from about 0.1 mg to about 35 mg daily, from about 0.1 mg toabout 30 mg daily, from about 0.1 mg to about 25 mg daily, from about0.1 mg to about 20 mg daily, from about 0.1 mg to about 15 mg daily,from about 0.1 mg to about 10 mg daily, from about 0.1 mg to about 5 mgdaily, from about 0.1 mg to about 3 mg daily, from about 0.1 mg to about1 mg daily, or from about 5 mg to about 80 mg daily. In variousembodiments, the chimeric protein is administered at a daily dose ofabout 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8mg, about 9 mg about 10 mg, about 15 mg, about 20 mg, about 25 mg, about30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg,about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about85 mg, about 90 mg, about 95 mg, about 100 mg, about 200 mg, about 500mg, about 1 g, about 2.5 g, about 5 g, about 7.5 g, about 10 g, about 25g, about 50 g, about 75 g, about 100 g, inclusive of all values andranges therebetween.

In accordance with certain embodiments of the invention, thepharmaceutical composition comprising the chimeric protein may beadministered, for example, more than once daily (e.g., about two times,about three times, about four times, about five times, about six times,about seven times, about eight times, about nine times, or about tentimes daily), about once per day, about every other day, about everythird day, about once a week, about once every two weeks, about onceevery month, about once every two months, about once every three months,about once every six months, or about once every year.

Combination Therapy and Additional Therapeutic Agents

In various embodiments, the pharmaceutical composition of the presentinvention is co-administered in conjunction with additional therapeuticagent(s). Co-administration can be simultaneous or sequential.

In one embodiment, the additional therapeutic agent and the chimericprotein of the present invention are administered to a subjectsimultaneously. The term “simultaneously” as used herein, means that theadditional therapeutic agent and the chimeric protein are administeredwith a time separation of no more than about 60 minutes, such as no morethan about 30 minutes, no more than about 20 minutes, no more than about10 minutes, no more than about 5 minutes, or no more than about 1minute. Administration of the additional therapeutic agent and thechimeric protein can be by simultaneous administration of a singleformulation (e.g., a formulation comprising the additional therapeuticagent and the chimeric protein) or of separate formulations (e.g., afirst formulation including the additional therapeutic agent and asecond formulation including the chimeric protein).

Co-administration does not require the therapeutic agents to beadministered simultaneously, if the timing of their administration issuch that the pharmacological activities of the additional therapeuticagent and the chimeric protein overlap in time, thereby exerting acombined therapeutic effect. For example, the additional therapeuticagent and the chimeric protein can be administered sequentially. Theterm “sequentially” as used herein means that the additional therapeuticagent and the chimeric protein are administered with a time separationof more than about 60 minutes. For example, the time between thesequential administration of the additional therapeutic agent and thechimeric protein can be more than about 60 minutes, more than about 2hours, more than about 5 hours, more than about 10 hours, more thanabout 1 day, more than about 2 days, more than about 3 days, more thanabout 1 week apart, more than about 2 weeks apart, or more than aboutone month apart. The optimal administration times will depend on therates of metabolism, excretion, and/or the pharmacodynamic activity ofthe additional therapeutic agent and the chimeric protein beingadministered. Either the additional therapeutic agent or the chimericprotein cell may be administered first.

Co-administration also does not require the therapeutic agents to beadministered to the subject by the same route of administration. Rather,each therapeutic agent can be administered by any appropriate route, forexample, parenterally or non-parenterally.

In some embodiments, the chimeric protein described herein actssynergistically when co-administered with another therapeutic agent. Insuch embodiments, the chimeric protein and the additional therapeuticagent may be administered at doses that are lower than the dosesemployed when the agents are used in the context of monotherapy.

In some embodiments, the present invention pertains to chemotherapeuticagents as additional therapeutic agents. For example, withoutlimitation, such combination of the present chimeric proteins andchemotherapeutic agent find use in the treatment of cancers, asdescribed elsewhere herein. Examples of chemotherapeutic agents include,but are not limited to, alkylating agents such as thiotepa and CYTOXANcyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(e.g., bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; cally statin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINdoxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as minoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOLpaclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANECremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), andTAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras,EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above. In addition, the methods of treatmentcan further include the use of radiation. In addition, the methods oftreatment can further include the use of photodynamic therapy.

In some embodiments, inclusive of, without limitation, infectiousdisease applications, the present invention pertains to anti-infectivesas additional therapeutic agents. In some embodiments, theanti-infective is an anti-viral agent including, but not limited to,Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir,Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir,Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, and Foscarnet. Insome embodiments, the anti-infective is an anti-bacterial agentincluding, but not limited to, cephalosporin antibiotics (cephalexin,cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole,cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics(cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracyclineantibiotics (tetracycline, minocycline, oxytetracycline, anddoxycycline); penicillin antibiotics (amoxicillin, ampicillin,penicillin V, dicloxacillin, carbenicillin, vancomycin, andmethicillin); monobactam antibiotics (aztreonam); and carbapenemantibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem).In some embodiments, the anti-infectives include anti-malarial agents(e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline,artemether/lumefantrine, atovaquone/proguanil andsulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin,pyrantel pamoate, and albendazole.

In some embodiments, inclusive, without limitation, of autoimmuneapplications, the additional therapeutic agent is an immunosuppressiveagent. In some embodiments, the immunosuppressive agent is ananti-inflammatory agent such as a steroidal anti-inflammatory agent or anon-steroidal anti-inflammatory agent (NSAID). Steroids, particularlythe adrenal corticosteroids and their synthetic analogues, are wellknown in the art. Examples of corticosteroids useful in the presentinvention include, without limitation, hydroxyltriamcinolone,alpha-methyl dexamethasone, beta-methyl betamethasone, beclomethasonedipropionate, betamethasone benzoate, betamethasone dipropionate,betamethasone valerate, clobetasol valerate, desonide, desoxymethasone,dexamethasone, diflorasone diacetate, diflucortolone valerate,fluadrenolone, fluclorolone acetonide, flumethasone pivalate,fluosinolone acetonide, fluocinonide, flucortine butylester,fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone,halcinonide, hydrocortisone acetate, hydrocortisone butyrate,methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone,flucetonide, fludrocortisone, difluorosone diacetate, fluradrenoloneacetonide, medrysone, amcinafel, amcinafide, betamethasone and thebalance of its esters, chloroprednisone, clocortelone, clescinolone,dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone,fluperolone, fluprednisolone, hydrocortisone, meprednisone,paramethasone, prednisolone, prednisone, beclomethasone dipropionate.(NSAIDS) that may be used in the present invention, include but are notlimited to, salicylic acid, acetyl salicylic acid, methyl salicylate,glycol salicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid,ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone,and indomethacin. In some embodiments, the immunosupressive agent may becytostatics such as alkylating agents, antimetabolites (e.g.,azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g.,basiliximab, daclizumab, and muromonab), anti-immunophilins (e.g.,cyclosporine, tacrolimus, sirolimus), inteferons, opioids, TNF bindingproteins, mycophenolates, and small biological agents (e.g., fingolimod,myriocin). Additional anti-inflammatory agents are described, forexample, in U.S. Pat. No. 4,537,776, the entire contents of which isincorporated by reference herein.

In some embodiments, the present invention pertains to various agentsused for treating obesity as additional therapeutic agents. Illustrativeagents used for treating obesity include, but are not limited to,orlistat (e.g. ALL1, XENICAL), loracaserin (e.g. BELVIQ),phentermine-topiramate (e.g. QSYMIA), sibutramme (e.g. REDUCTIL orMERJDIA), rimonabant (ACOMPLLA), exenatide (e.g. BYETTA), pramlintide(e.g. SYMLIN) phentermine, benzphetamine, diethylpropion,phendimetrazme, bupropion, and metformin. Agents that interfere with thebody's ability to absorb specific nutrients in food are among theadditional agents, e.g. orlistat (e.g. ALU, XENICAL), glucomannan, andguar gum. Agents that suppress apetite are also among the additionalagents, e.g. catecholamines and their derivatives (such as phenteimineand other amphetamine-based drugs), various antidepressants and moodstabilizers (e.g. bupropion and topiramate), anorectics (e.g. dexedrine,digoxin). Agents that increase the body's metabolism are also among theadditional agents.

In some embodiments, additional therapeutic agents may be selected fromamong appetite suppressants, neurotransmitter reuptake inhibitors,dopaminergic agonists, serotonergic agonists, modulators of GABAergicsignaling, anticonvulsants, antidepressants, monoamine oxidaseinhibitors, substance P (NK1) receptor antagonists, melanocortinreceptor agonists and antagonists, lipase inhibitors, inhibitors of fatabsorption, regulators of energy intake or metabolism, cannabinoidreceptor modulators, agents for treating addiction, agents for treatingmetabolic syndrome, peroxisome proliferator-activated receptor (PPAR)modulators; dipcptidyl peptidase 4 (DPP-4) antagonists, agents fortreating cardiovascular disease, agents for treating elevatedtriglyceride levels, agents for treating low HDL, agents for treatinghypercholesterolemia, and agents for treating hypertension. Some agentsfor cardiovascular disease include statins (e.g. lovastatin,atorvastatin, fluvastatin, rosuvastatin, simvastatin and pravastatin)and omega-3 agents (e.g. LOVAZA, EPANQVA, VASCEPA, esterified omega-3'sin general, fish oils, krill oils, algal oils). In some embodiments,additional agents may be selected from among amphetamines,benzodiazepines, sulfonyl ureas, meglitinides, thiazolidinediones,biguanides, beta-blockers, XCE inhibitors, diuretics, nitrates, calciumchannel blockers, phenlermine, sibutramine, iorcaserin, cetilistat,rimonabant, taranabant, topiramate, gabapentin, valproate, vigabatrin,bupropion, tiagabine, sertraline, fluoxetine, trazodone, zonisamide,methylphenidate, varenicline, naltrexone, diethylpropion,phendimetrazine, rcpaglini.de, nateglinide, glimepiride, metformin,pioglitazone, rosiglilazone, and sitagliptin.

In some embodiments, the present invention pertains to an agent used fortreating diabetes as additional therapeutic agents. Illustrativeanti-diabetic agents include, but are not limited to, sulfonylurea (e.g.DYMELOR (acetohexamide), DIABINESE (chlorpropamide), ORINASE(tolbutamide), and TOLINASE (tolazamide), GLUCOTROL (glipizide),GLUCOTROL XL (extended release), DIABETA (glyburide), MICRONASE(glyburide), GLYNASE PRESTAB (glyburide), and AMARYL (glimepiride)); aBiguanide (e.g. metformin (GLUCOPHAGE, GLUCOPHAGE XR, RIOMET, FORTAMET,and GLUMETZA)); a thiazolidinedione (e.g. ACTOS (pioglitazone) andAVANDIA (rosiglitazone); an alpha-glucosidase inhibitor (e.g., PRECOSE(acarbose) and GLYSET (miglitol); a Meglitinide (e.g., PRANDIN(repaglinide) and STARLIX (nateglinide)); a Dipeptidyl peptidase IV(DPP-IV) inhibitor (e.g., JANUVIA (sitagliptin), NESINA (alogliptin),ONGLYZA (saxagliptin), and TRADJENTA (linagliptin)); Sodium-glucoseco-transporter 2 (SGLT2) inhibitor (e.g. INVOKANA (canaglifozin)); and acombination pill (e.g. GLUCOVANCE, which combines glyburide (asulfonylurea) and metformin, METAGLIP, which combines glipizide (asulfonylurea) and metformin, and AVANDAMET, which uses both metforminand rosiglitazone (AVANDIA) in one pill, KAZANO (alogliptin andmetformin), OSENI (alogliptin plus pioglitazone), METFORMIN oral, ACTOSoral, BYETTA subcutaneous, JANUVIA oral, WELCHOL oral, JANUMET oral,glipizide oral, glimepiride oral, GLUCOPHAGE oral, LANTUS subcutaneous,glyburide oral, ONGLYZA oral, AMARYI oral, LANTUS SOLOSTAR subcutaneous,BYDUREON subcutaneous, LEVEMIR FLEXPEN subcutaneous, ACTOPLUS MET oral,GLUMETZA oral, TRADJENTA oral, bromocriptine oral, KOMBIGLYZE XR oral,INVOKANA oral, PRANDIN oral, LEVEMIR subcutaneous, PARLODEL oral,pioglitazone oral, NOVOLOG subcutaneous, NOVOLOG FLEXPEN subcutaneous,VICTOZA 2-PAK subcutaneous, HUMALOG subcutaneous, STARLIX oral, FORTAMEToral, GLUCOVANCE oral, GLUCOPHAGE XR oral, NOVOLOG Mix 70-30 FLEXPENsubcutaneous, GLYBURIDE-METFORMIN oral, acarbose oral, SYMLINPEN 60subcutaneous, GLUCOTROI XL oral, NOVOLIN R inj, GLUCOTROL oral, DUETACToral, sitagliptin oral, SYMLINPEN 120 subcutaneous, HUMALOG KWIKPENsubcutaneous, JANUMET XR oral, GLIPIZIDE-METFORMIN oral, CYCLOSET oral,HUMALOG MIX 75-25 subcutaneous, nateglinide oral, HUMALOG Mix 75-25KWIKPEN subcutaneous, HUMULIN 70/30 subcutaneous, PRECOSE oral, APIDRAsubcutaneous, Humulin R inj, Jentadueto oral, Victoza 3-Paksubcutaneous, Novolin 70/30 subcutaneous, NOVOLIN N subcutaneous,insulin detemir subcutaneous, glyburide micronized oral, GLYNASE oral,HUMULIN N subcutaneous, insulin glargine subcutaneous, RIOMET oral,pioglitazone-metformin oral, APIDRA SOLOSTAR subcutaneous, insulinlispro subcutaneous, GLYSET oral, HUMULIN 70/30 Pen subcutaneous,colesevelam oral, sitagliptin-metformin oral, DIABETA oral, insulinregular human inj, HUMULIN N Pen subcutaneous, exenatide subcutaneous,HUMALOG Mix 50-50 KWIKPEN subcutaneous, liraglutide subcutaneous, KAZANOoral, repaglinide oral, chlorpropamide oral, insulin aspartsubcutaneous, NOVOLOG Mix 70-30 subcutaneous, HUMALOG Mix 50-50subcutaneous, saxagliptin oral, ACTOPLUS Met XR oral, miglitol oral, NPHinsulin human recomb subcutaneous, insulin NPH and regular humansubcutaneous, tolazamide oral, mifepristone oral, insulin aspartprotam-insulin aspart subcutaneous, repaglinide-metformin oral,saxagliptin-metformin oral, linagliptin-metformin oral, NESINA oral,OSENI oral, tolbutamide oral, insulin lispro protamine and lisprosubcutaneous, pramlintide subcutaneous, insulin glulisine subcutaneous,pioglitazone-glimepiride oral, PRANDIMET oral, NOVOLOG PenFillsubcutaneous, linagliptin oral, exenatide microspheres subcutaneous,KORLYM oral, alogliptin oral, alogliptin-pioglitazone oral,alogliptin-metformin oral, canagliflozin oral, Lispro (HUMALOG); Aspart(NOVOLOG); Glulisine (APIDRA); Regular (NOVOLIN R or HUMULIN R); NPH(NOVOLIN N or HUMULIN N); Glargine (LANTUS); Detemir (LEVEMIR); HUMULINor NOVOLIN 70/30; and NOVOLOG Mix 70/30 HUMALOG Mix 75/25 or 50/50.

In some embodiments, the present invention pertains to an agent used fortreating MS as additional therapeutic agents. Illustrative MS agentsinclude, but are not limited to various disease modifying therapies:

Generic Name Branded Name/Company Frequency/Route of Delivery/Usual Doseteriflunomide AUBAGIO (GENZYME) Every day; pill taken orally; 7 mg or 14mg. interferon beta-1a AVONEX (BIOGEN IDEC) Once a week; intramuscular(into the muscle) injection; 30 mcg interferon beta-1b BETASERON (BAYEREvery other day; subcutaneous (under the skin) HEALTHCARE injection; 250mcg. PHARMACEUTICALS, INC.) glatiramer acetate COPAXONE (TEVA Every day;subcutaneous (under the skin) NEUROSCIENCE) injection; 20 mg (20,000mcg) OR Three times a week; subcutaneous (under the skin) injection; 40mg (40,000 mcg) interferon beta-1b EXTAVIA (NOVARTIS Every other day;subcutaneous (under the skin) PHARMACEUTICALS CORP.) injection; 250 mcg.fingolimod GILENYA (NOVARTIS Every day; capsule taken orally; 0.5 mg.PHARMACEUTICALS CORP.) Alemtuzumab (anti-CD52 LEMTRADA (GENZYME)Intravenous infusion on five consecutive days, monoclonal antibody)followed by intravenous infusion on three consecutive days one yearlater (12 mg) mitoxantrone NOVANTRONE (EMD Four times a year by IVinfusion in a medical SERONO) facility. Lifetime cumulative dose limitof approximately 8-12 doses over 2-3 years (140 mg/m2). pegylatedinterferon beta-1a PLEGRIDY (BIOGEN IDEC) Every 14 days; subcutaneous(under the skin) injection; 125 mcg interferon beta-1a REBIF (EMDSERONO, INC.) Three times a week; subcutaneous (under the skin)injection; 44 mcg dimethyl fumarate (BG-12) TECFIDERA (BIOGEN IDEC)Twice a day; capsule taken orally; 120 mg for one week and 240 mgtherafter Natalizumab (humanized TYSABRI (BIOGEN IDEC) Every four weeksby IV infusion in a registered monoclonal antibody VLA-4 infusionfacility; 300 mg antagonist) DMTs in Development Amiloride (targetsAcid- PAR PHARMACEUTICAL, Oral sensing ion channel-1 PERRIGO COMPANY,Epithelial sodium channel SIGMAPHARM Na+/H+ exchanger) LABORATORIESATX-MS-1467 (targets Major APITOPE/MERCK SERONO Intradermal Subcutaneoushistocompatibility complex class II T cell responses to myelin basicprotein) BAF312 (targets NOVARTIS PHARMA Oral Sphingosine 1-phosphate(S1P) receptor subtypes S1P1 and S1P5 B cell distrubution T celldistribution) BGC20-0134 (targets BTG PLC Oral Proinflammatory and anti-inflammatory cytokines) BIIB033 (targets LINGO-1 BIOGEN Intravenousinfusion used in Phase I and Phase II (“leucine-rich repeat and trialsSubcutaneous injection used in Phase I trial immunoglobulin-like domain-containing, Nogo receptor- interacting protein”)) Cladribine (targetsCD4+T MERCK SERONO Oral cells DNA synthesis and repair E-selectinIntracellular adhesion molecule-1 Pro- inflammatory cytokinesinterleukin 2 and interleukin 2R Pro-inflammatory cytokines interleukin8 and RANTES Cytokine secretion Monocyte and lymphocyte migration)Cyclophosphamide (targets BAXTER HEALTHCARE Oral, monthly intravenouspulses T cells, particularly CD4+ CORPORATION helper T cells B cells)Daclizumab (humanized BIOGEN IDEC/ABBVIE Projected to be IM injectiononce monthly monoclonal antibody BIOTHERAPEUTICS targeting CD25 Immunemodulator of T cells) Dalfampridine (targets ACORDA THERAPEUTICS/ Onetablet every 12 hours (extended release), 10 Voltage-gated potassiumBIOGEN IDEC mg twice a day channels Degenerin/epithelial sodium channelsL-type calcium channels that contain subunit Cavbeta3) Dronabinol(targets ABBVIE INC. Oral Cannabinoid receptor CB1 Cannabinoid receptorCB2) Firategrast (targets GLAXOSMITHKLINE Oral Alpha4beta1 integrin)GNbAC1MSRV-Env (targets GENEURO SA/SERVIER Intravenous infusion envelopeprotein of the MS- associated retrovirus) Idebenone (targets ReactiveSANTHERA Oral Dose in clinical trial for PPMS is 2250 mg per oxygenspecies) PHARMACEUTICALS day (750 mg dose, 3 times per day)Imilecleucel-T (targets OPEM THERAPEUTICS/ Subcutaneous Given 5 timesper year, according Myelin-specific, autoreactive MERCK SERONO toinformation from the manufacturer T cells) Laquinimod TEVA Projected tobe 0.6 mg or 1.2 mg oral tablet taken daily Masitinib (targets KIT (a ABSCIENCE Oral stem cell factor, also called c-KIT) receptor as well asselect other tyrosine kinases Mast cells) MEDI-551 (targets CD19, aMEDIMMUNE Intravenous Subcutaneous B cell-specific antigen that is partof the B cell receptor complex and that functions in determining thethreshold for B cell activation B cells Plasmablasts, B cells thatexpress CD19 (but not CD20) and that secrete large quantities ofantibodies; depletion of plasmablasts may be useful in autoimmunediseases involving pathogenic autoantibodies) Minocycline (targets Tcells VARIOUS Oral Available as pellet-filled capsules and an oralMicroglia Leukocyte suspension migration Matrix metalloproteinases)MIS416 (targets Innate INNATE Intravenous immune system Pathogen-IMMUNOTHERAPEUTICS associated molecular pattern recognition receptors ofthe innate immune system Myeloid cells of the innate immune system,which might be able to remodel the deregulated immune system activitythat occurs in SPMS) Mycophenolate mofetil MANUFACTURED BY Oral (targetsPurine synthesis) GENENTECH Naltrexone (targets Opioid VARIOUS Given atlow doses (3 to 4.5 mg per day) in oral receptors Toll-like receptorform as “Low-dose naltrexone” (or “LDN”) 4) Ocrelizumab and ROCHE/GSKProjected to be IV infusion Ofatumumab (humanized monoclonal antibodiestargeting CD20 B cell suppression ONO-4641 (targets ONO PHARMACEUTICALCO. Oral Sphingosine 1-phosphate receptor) Phenytoin (targets SodiumPFIZER Intravenous Intramuscular (less favored option) channels) OralPonesimod ACTELION To be determined Raltegravir (targets MERCK Oral 400mg tablet twice daily, according to Retroviral integrase informationfrom the manufacturer Herpesvirus DNA packaging terminase) RHB 104REDHILL BIOPHARMA 95 mg clarithromycin, 45 mg rifabutin, and 10 mgLIMITED clofazimine Riluzole (targets COVIS PHARMA/SANOFI OralGlutamatergic neurotransmission Glutamate uptake and releaseVoltage-gated sodium channels Protein kinase C)

In some embodiments, the present invention relates to combinationtherapy with a blood transfusion. For instance, the present compositionsmay supplement a blood transfusion. In some embodiments, the presentinvention relates to combination therapy with iron supplements.

In some embodiments, the present invention relates to combinationtherapy with one or more EPO-based agents. For example, the presentcompositions may be used as an adjuvant to other EPO-based agents. Insome embodiments, the present compositions are used as a maintenancetherapy to other EPO-based agents. Other EPO-based agents include thefollowing: epoetin alfa, including without limitation, DARBEPOETIN(ARANESP), EPOCEPT (LUPIN PHARMA), NANOKINE (NANOGEN PHARMACEUTICAL),EPOFIT (INTAS PHARMA), EPOGEN (AMGEN), EPOGIN, EPREX, (JANSSEN-CILAG),BINOCRIT7 (SANDOZ), PROCRIT; epoetin beta, including without limitation,NEORECORMON (HOFFMANN-LA ROCHE), RECORMON, Methoxy polyethyleneglycol-epoetin beta (MIRCERA, ROCHE); epoetin delta, including withoutlimitation, DYNEPO (erythropoiesis stimulating protein, SHIRE PLC);epoetin omega, including without limitation, EPOMAX; epoetin zeta,including without limitation, SILAPO (STADA) and RETACRIT (HOSPIRA) andother EPOs, including without limitation, EPOCEPT (LUPINPHARMACEUTICALS), EPOTRUST (PANACEA BIOTEC LTD), ERYPRO SAFE (BIOCONLTD.), REPOITIN (SERUM INSTITUTE OF INDIA LIMITED), VINTOR (EMCUREPHARMACEUTICALS), EPOFIT (INTAS PHARMA), ERYKINE (INTASBIOPHARMACEUTICA), WEPOX (WOCKHARDT BIOTECH), ESPOGEN (LG LIFESCIENCES), RELIPOIETIN (RELIANCE LIFE SCIENCES), SHANPOIETIN (SHANTHABIOTECHNICS LTD), ZYROP (CADILA HEALTHCARE LTD.), EPIAO (RHUEPO)(SHENYANG SUNSHINE PHARMACEUTICAL CO. LTD), CINNAPOIETIN (CINNAGEN).

In some embodiments, the present invention relates to combinationtherapy with one or more immune-modulating agents, for example, withoutlimitation, agents that modulate immune checkpoint. In variousembodiments, the immune-modulating agent targets one or more of PD-1,PD-L1, and PD-L2. In various embodiments, the immune-modulating agent isPD-1 inhibitor. In various embodiments, the immune-modulating agent isan antibody specific for one or more of PD-1, PD-L1, and PD-L2. Forinstance, in some embodiments, the immune-modulating agent is anantibody such as, by way of non-limitation, nivolumab,(ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB),pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH),MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL328OA (ROCHE).In some embodiments, the immune-modulating agent targets one or more ofCD137 or CD137L. In various embodiments, the immune-modulating agent isan antibody specific for one or more of CD137 or CD137L. For instance,in some embodiments, the immune-modulating agent is an antibody such as,by way of non-limitation, urelumab (also known as BMS-663513 andanti-4-1BB antibody). In some embodiments, the present chimeric proteinis combined with urelumab (optionally with one or more of nivolumab,lirilumab, and urelumab) for the treatment of solid tumors and/or B-cellnon-Hodgkins lymphoma and/or head and neck cancer and/or multiplemyeloma. In some embodiments, the immune-modulating agent is an agentthat targets one or more of CTLA-4, AP2M1, CD80, CD86, SHP-2, andPPP2R5A. In various embodiments, the immune-modulating agent is anantibody specific for one or more of CTLA-4, AP2M1, CD80, CD86, SHP-2,and PPP2R5A. For instance, in some embodiments, the immune-modulatingagent is an antibody such as, by way of non-limitation, ipilimumab(MDX-010, MDX-101, Yervoy, BMS) and/or tremelimumab (Pfizer). In someembodiments, the present chimeric protein is combined with ipilimumab(optionally with bavituximab) for the treatment of one or more ofmelanoma, prostate cancer, and lung cancer. In various embodiments, theimmune-modulating agent targets CD20. In various embodiments, theimmune-modulating agent is an antibody specific CD20. For instance, insome embodiments, the immune-modulating agent is an antibody such as, byway of non-limitation, Ofatumumab (GENMAB), obinutuzumab (GAZYVA),AME-133v (APPLIED MOLECULAR EVOLUTION), Ocrelizumab (GENENTECH), TRU-015(TRUBION/EMERGENT), veltuzumab (IMMU-106).

In some embodiments, the present invention relates to combinationtherapy with one or more chimeric agents described in WO 2013/10779, WO2015/007536, WO 2015/007520, WO 2015/007542, and WO 2015/007903, theentire contents of which are hereby incorporated by reference in theirentireties.

In some embodiments, the chimeric protein described herein, includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the composition such that covalent attachment doesnot prevent the activity of the composition. For example, but not by wayof limitation, derivatives include composition that have been modifiedby, inter alia, glycosylation, lipidation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications can be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc.

In still other embodiments, the chimeric protein described hereinfurther comprise a cytotoxic agent, comprising, in illustrativeembodiments, a toxin, a chemotherapeutic agent, a radioisotope, and anagent that causes apoptosis or cell death. Such agents may be conjugatedto a composition described herein.

The chimeric protein described herein may thus be modifiedpost-translationally to add effector moieties such as chemical linkers,detectable moieties such as for example fluorescent dyes, enzymes,substrates, bioluminescent materials, radioactive materials, andchemiluminescent moieties, or functional moieties such as for examplestreptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, andradioactive materials.

Illustrative cytotoxic agents include, but are not limited to,methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine; alkylating agents such as mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C,lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide,mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C,cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin(paraplatin); anthracyclines include daunorubicin (formerly daunomycin),doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin,epirubicin, mitoxantrone and bisantrene; antibiotics includedactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, andanthramycin (AMC); and antimytotic agents such as the vinca alkaloids,vincristine and vinblastine. Other cytotoxic agents include paclitaxel(taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B,gramicidin D, ethidium bromide, emetine, etoposide, tenoposide,colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids,mytotane (O,P′-(DDD)), interferons, and mixtures of these cytotoxicagents.

Further cytotoxic agents include, but are not limited to,chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel,gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C,actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGFantagonists, EGFR antagonists, platins, taxols, irinotecan,5-fluorouracil, gemcytabine, leucovorine, steroids, cyclophosphamide,melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesineand vinorelbine), mustines, tyrosine kinase inhibitors, radiotherapy,sex hormone antagonists, selective androgen receptor modulators,selective estrogen receptor modulators, PDGF antagonists, TNFantagonists, IL-1 antagonists, interleukins (e.g. IL-12 or IL-2), IL-12Rantagonists, Toxin conjugated monoclonal antibodies, tumor antigenspecific monoclonal antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20antibodies, Rituxan, ocrelizumab, ofatumumab, DXL625, HERCEPTIN®, or anycombination thereof. Toxic enzymes from plants and bacteria such asricin, diphtheria toxin and Pseudomonas toxin may be conjugated to thetherapeutic agents (e.g. antibodies) to generatecell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad.Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419(1980)).

Other cytotoxic agents include cytotoxic ribonucleases as described byGoldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention alsorelate to radioimmunoconjugates where a radionuclide that emits alpha orbeta particles is stably coupled to the chimeric protein, with orwithout the use of a complex-forming agent. Such radionuclides includebeta-emitters such as Phosphorus-32, Scandium-47, Copper-67, Gallium-67,Yttrium-88, Yttrium-90, Iodine-125, Iodine-131, Samarium-153,Lutetium-177, Rhenium-186 or Rhenium-188, and alpha-emitters such asAstatine-211, Lead-212, Bismuth-212, Bismuth-213 or Actinium-225.

Illustrative detectable moieties further include, but are not limitedto, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase,beta-galactosidase and luciferase. Further illustrative fluorescentmaterials include, but are not limited to, rhodamine, fluorescein,fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine,phycoerythrin and dansyl chloride. Further illustrative chemiluminescentmoieties include, but are not limited to, luminol. Further illustrativebioluminescent materials include, but are not limited to, luciferin andaequorin. Further illustrative radioactive materials include, but arenot limited to, Iodine-125, Carbon-14, Sulfur-35, Tritium andPhosphorus-32.

Methods of Treatment

Methods and compositions described herein have application to treatingvarious diseases and disorders, including, but not limited to cancer,infections, immune disorders, anemia, autoimmune diseases,cardiovascular diseases, wound healing, ischemia-related diseases,neurodegenerative diseases, metabolic diseases and many other diseasesand disorders.

Further, any of the present agents may be for use in the treating, orthe manufacture of a medicament for treating, various diseases anddisorders, including, but not limited to cancer, infections, immunedisorders, inflammatory diseases or conditions, and autoimmune diseases.

In some embodiments, the present invention relates to the treatment of,or a patient having one or more of cancer, heart failure, autoimmunedisease, sickle cell disease, thalassemia, blood loss, transfusionreaction, diabetes, vitamin B12 deficiency, collagen vascular disease,Shwachman syndrome, thrombocytopenic purpura, Celiac disease, endocrinedeficiency state such as hypothyroidism or Addison's disease, autoimmunedisease such as Crohn's Disease, systemic lupus erythematosis,rheumatoid arthritis or juvenile rheumatoid arthritis, ulcerativecolitis immune disorders such as eosinophilic fasciitis,hypoimmunoglobulinemia, or thymoma/thymic carcinoma, graft versus hostdisease, preleukemia, Nonhematologic syndrome (e.g. Down's, Dubowwitz,Seckel), Felty syndrome, hemolytic uremic syndrome, myelodysplasicsyndrome, nocturnal paroxysmal hemoglobinuria, osteomyelofibrosis,pancytopenia, pure red-cell aplasia, Schoenlein-Henoch purpura, malaria,protein starvation, menorrhagia, systemic sclerosis, liver cirrhosis,hypometabolic states, and congestive heart failure.

In some embodiments, the present invention relates to the treatment of,or a patient having cancer. As used herein, cancer refers to anyuncontrolled growth of cells that may interfere with the normalfunctioning of the bodily organs and systems, and includes both primaryand metastatic tumors. Primary tumors or cancers that migrate from theiroriginal location and seed vital organs can eventually lead to the deathof the subject through the functional deterioration of the affectedorgans. A metastasis is a cancer cell or group of cancer cells, distinctfrom the primary tumor location, resulting from the dissemination ofcancer cells from the primary tumor to other parts of the body.Metastases may eventually result in death of a subject. For example,cancers can include benign and malignant cancers, polyps, hyperplasia,as well as dormant tumors or micrometastases.

Illustrative cancers that may be treated include, but are not limitedto, carcinomas, e.g. various subtypes, including, for example,adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, andtransitional cell carcinoma), sarcomas (including, for example, bone andsoft tissue), leukemias (including, for example, acute myeloid, acutelymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell),lymphomas and myelomas (including, for example, Hodgkin and non-Hodgkinlymphomas, light chain, non-secretory, MGUS, and plasmacytomas), andcentral nervous system cancers (including, for example, brain (e.g.gliomas (e.g. astrocytoma, oligodendroglioma, and ependymoma),meningioma, pituitary adenoma, and neuromas, and spinal cord tumors(e.g. meningiomas and neurofibroma).

Illustrative cancers that may be treated include, but are not limitedto, basal cell carcinoma, biliary tract cancer; bladder cancer; bonecancer; brain and central nervous system cancer; breast cancer; cancerof the peritoneum; cervical cancer; choriocarcinoma; colon and rectumcancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm;kidney or renal cancer; larynx cancer; leukemia; liver cancer; lungcancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue,mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as wellas B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (e.g. that associated with brain tumors), andMeigs' syndrome.

In some embodiments, the present invention relates to the treatment of,or a patient having a microbial infection and/or chronic infection.Illustrative infections include, but are not limited to, HIV/AIDS,tuberculosis, osteomyelitis, hepatitis B, hepatitis C, Epstein-Barrvirus or parvovirus, T cell leukemia virus, bacterial overgrowthsyndrome, fungal or parasitic infections.

In various embodiments, the present compositions are used to treat orprevent one or more inflammatory diseases or conditions, such asinflammation, acute inflammation, chronic inflammation, respiratorydisease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopicdermatitis, septic shock, rheumatoid arthritis, inflammatory boweldisease, inflammatory pelvic disease, pain, ocular inflammatory disease,celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familialeosinophilia (FE), autosomal recessive spastic ataxia, laryngealinflammatory disease; Tuberculosis, Chronic cholecystitis,Bronchiectasis, Silicosis and other pneumoconioses.

In various embodiments, the present compositions are used to treat orprevent one or more autoimmune diseases or conditions, such as multiplesclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease,ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture'ssyndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen'sencephalitis, Primary biliary sclerosis, Sclerosing cholangitis,Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis,Fibromyalgia, Menier's syndrome; transplantation rejection (e.g.,prevention of allograft rejection) pernicious anemia, rheumatoidarthritis, systemic lupus erythematosus, dermatomyositis, Sjogren'ssyndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis,Reiter's syndrome, Grave's disease, and other autoimmune diseases.

In various embodiments, the present compositions are used to treat,control or prevent cardiovascular disease, such as a disease orcondition affecting the heart and vasculature, including but not limitedto, coronary heart disease (CHD), cerebrovascular disease (CVD), aorticstenosis, peripheral vascular disease, atherosclerosis,arteriosclerosis, myocardial infarction (heart attack), cerebrovasculardiseases (stroke), transient ischemic attacks (TIA), angina (stable andunstable), atrial fibrillation, arrhythmia, vascular disease, and/orcongestive heart failure.

In various embodiments, the present compositions are used to treat orprevent one or more metabolic-related disorders. In various embodiments,the present invention is useful for the treatment, controlling orprevention of diabetes, including Type 1 and Type 2 diabetes anddiabetes associated with obesity. The compositions and methods of thepresent invention are useful for the treatment or prevention ofdiabetes-related disorders, including without limitation diabeticnephropathy, hyperglycemia, impaired glucose tolerance, insulinresistance, obesity, lipid disorders, dyslipidemia, hyperlipidemia,hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDLlevels, atherosclerosis and its sequelae, vascular restenosis, irritablebowel syndrome, inflamatory bowel disease, including Crohn's disease andulcerative colitis, other inflammatory conditions, pancreatitis,abdominal obesity, neurodegenerative disease, retinopathy, neoplasticconditions, adipose cell tumors, adipose cell carcinomas, such asliposarcoma, prostate cancer and other cancers, including gastric,breast, bladder and colon cancers, angiogenesis, Alzheimer's disease,psoriasis, high blood pressure, Metabolic Syndrome (e.g. a person hasthree or more of the following disorders: abdominal obesity,hypertriglyceridemia, low HDL cholesterol, high blood pressure, and highfasting plasma glucose), ovarian hyperandrogenism (polycystic ovarysyndrome), and other disorders where insulin resistance is a component,such as sleep apnea. The compositions and methods of the presentinvention are useful for the treatment, control, or prevention ofobesity, including genetic or environmental, and obesity-relateddisorders. The obesity-related disorders herein are associated with,caused by, or result from obesity. Examples of obesity-related disordersinclude obesity, diabetes, overeating, binge eating, and bulimia,hypertension, elevated plasma insulin concentrations and insulinresistance, dyslipidemia, hyperlipidemia, endometrial, breast, prostate,kidney and colon cancer, osteoarthritis, obstructive sleep apnea,gallstones, heart disease, abnormal heart rhythms and arrythmias,myocardial infarction, congestive heart failure, coronary heart disease,sudden death, stroke, polycystic ovary disease, craniopharyngioma,Prader-Willi Syndrome, Frohlich's syndrome, GH-deficient subjects,normal variant short stature, Turner's syndrome, and other pathologicalconditions showing reduced metabolic activity or a decrease in restingenergy expenditure as a percentage of total fat-free mass, e.g, childrenwith acute lymphoblastic leukemia. Further examples of obesity-relateddisorders are Metabolic Syndrome, insulin resistance syndrome,reproductive hormone abnormalities, sexual and reproductive dysfunction,such as impaired fertility, infertility, hypogonadism in males andhirsutism in females, fetal defects associated with maternal obesity,gastrointestinal motility disorders, such as obesity-relatedgastro-esophageal reflux, respiratory disorders, such asobesity-hypoventilation syndrome (Pickwickian syndrome), breathlessness,cardiovascular disorders, inflammation, such as systemic inflammation ofthe vasculature, arteriosclerosis, hypercholesterolemia, lower backpain, gallbladder disease, hyperuricemia, gout, and kidney cancer, andincreased anesthetic risk. The compositions and methods of the presentinvention are also useful to treat Alzheimer's disease.

In various embodiments, the present compositions are used to treat orprevent one or more respiratory diseases, such as asthma, chronicobstructive pulmonary disease (COPD), bronchiectasis, allergic rhinitis,sinusitis, pulmonary vasoconstriction, inflammation, allergies, impededrespiration, respiratory distress syndrome, cystic fibrosis, pulmonaryhypertension, pulmonary vasoconstriction, emphysema, Hantaviruspulmonary syndrome (HPS), Loeffler's syndrome, Goodpasture's syndrome,Pleurisy, pneumonitis, pulmonary edema, pulmonary fibrosis, Sarcoidosis,complications associated with respiratory syncitial virus infection, andother respiratory diseases.

In some embodiments, the present invention is used to treat or preventone or more neurodegenerative disease. Illustrative neurodegenerativedisease include, but are not limited to, multiple sclerosis (includingwithout limitation, benign multiple sclerosis; relapsing-remittingmultiple sclerosis (RRMS); secondary progressive multiple sclerosis(SPMS); progressive relapsing multiple sclerosis (PRMS); and primaryprogressive multiple sclerosis (PPMS)), Alzheimer's disease (including,without limitation, Early-onset Alzheimer's, Late-onset Alzheimer's, andFamilial Alzheimer's disease (FAD), Parkinson's disease and parkinsonism(including, without limitation, Idiopathic Parkinson's disease, Vascularparkinsonism, Drug-induced parkinsonism, Dementia with Lewy bodies,Inherited Parkinson's, Juvenile Parkinson's), Huntington's disease,Amyotrophic lateral sclerosis (ALS, including, without limitation,Sporadic ALS, Familial ALS, Western Pacific ALS, Juvenile ALS, HiramayaDisease).

In various embodiments, the present chimeric proteins find use intreating wounds, e.g., a non-healing wound, an ulcer, a burn, orfrostbite, a chronic or acute wound, open or closed wound, internal orexternal wound (illustrative external wounds are penetrating andnon-penetrating wound.

In various embodiments, the present chimeric proteins find use intreating ischemia, by way of non-limiting example, ischemia associatedwith acute coronary syndrome, acute lung injury (ALI), acute myocardialinfarction (AMI), acute respiratory distress syndrome (ARDS), arterialocclusive disease, arteriosclerosis, articular cartilage defect, asepticsystemic inflammation, atherosclerotic cardiovascular disease,autoimmune disease, bone fracture, bone fracture, brain edema, brainhypoperfusion, Buerger's disease, burns, cancer, cardiovascular disease,cartilage damage, cerebral infarct, cerebral ischemia, cerebral stroke,cerebrovascular disease, chemotherapy-induced neuropathy, chronicinfection, chronic mesenteric ischemia, claudication, congestive heartfailure, connective tissue damage, contusion, coronary artery disease(CAD), critical limb ischemia (CLI), Crohn's disease, deep veinthrombosis, deep wound, delayed ulcer healing, delayed wound-healing,diabetes (type I and type II), diabetic neuropathy, diabetes inducedischemia, disseminated intravascular coagulation (DIC), embolic brainischemia, frostbite, graft-versus-host disease, hereditary hemorrhagictelengiectasiaischemic vascular disease, hyperoxic injury, hypoxia,inflammation, inflammatory bowel disease, inflammatory disease, injuredtendons, intermittent claudication, intestinal ischemia, ischemia,ischemic brain disease, ischemic heart disease, ischemic peripheralvascular disease, ischemic placenta, ischemic renal disease, ischemicvascular disease, ischemic-reperfusion injury, laceration, left maincoronary artery disease, limb ischemia, lower extremity ischemia,myocardial infarction, myocardial ischemia, organ ischemia,osteoarthritis, osteoporosis, osteosarcoma, Parkinson's disease,peripheral arterial disease (PAD), peripheral artery disease, peripheralischemia, peripheral neuropathy, peripheral vascular disease,pre-cancer, pulmonary edema, pulmonary embolism, remodeling disorder,renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers,solid organ transplantation, spinal cord injury, stroke,subchondral-bone cyst, thrombosis, thrombotic brain ischemia, tissueischemia, transient ischemic attack (TIA), traumatic brain injury,ulcerative colitis, vascular disease of the kidney, vascularinflammatory conditions, von Hippel-Lindau syndrome, or wounds totissues or organs

In various embodiments, the present invention relates to the treatmentof one or more of anemia, including anemia resulting from chronic kidneydisease (e.g. from dialysis) and/or an anti-cancer agent (e.g.chemotherapy and/or HIV treatment (e.g. Zidovudine (INN) orazidothymidine (AZT)), inflammatory bowel disease (e.g. Crohn's diseaseand ulcer colitis), anemia linked to inflammatory conditions (e.g.arthritis, lupus, IBD), anemia linked to diabetes, schizophrenia,cerebral malaria, as aplastic anemia, and myelodysplasia from thetreatment of cancer (e.g. chemotherapy and/or radiation), and variousmyelodysplastic syndrome diseases (e.g. sickle cell anemia, hemoglobinSC disease, hemoglobin C disease, alpha- and beta-thalassemias, neonatalanemia after premature birth, and comparable conditions).

In some embodiments, the present invention relates to the treatment of,or a patient having anemia, i.e. a condition in which the number of redblood cells and/or the amount of hemoglobin found in the red blood cellsis below normal. In various embodiments, the anemia may be acute orchronic. For example, the present anemias include but are not limited toiron deficiency anemia, renal anemia, anemia of chronicdiseases/inflammation, pernicious anemia such as macrocytic achylicanemia, juvenile pernicious anemia and congenital pernicious anemia,cancer-related anemia, anti-cancer-related anemia (e.g.chemotherapy-related anemia, radiotherapy-related anemia), pure red cellaplasia, refractory anemia with excess of blasts, aplastic anemia,X-lined sideroblastic anemia, hemolytic anemia, sickle cell anemia,anemia caused by impaired production of ESA, myelodysplasia syndromes,hypochromic anemia, microcytic anemia, sideroblastic anemia, autoimmunehemolytic anemia, Cooley's anemia, Mediterranean anemia, DiamondBlackfan anemia, Fanconi's anemia and drug-induced immune hemolyticanemia. Anemia may cause serious symptoms, including hypoxia, chronicfatigue, lack of concentration, pale skin, low blood pressure, dizzinessand heart failure.

In some embodiments, the present invention relates to the treatment ofanemia resulting from chronic renal failure. In some embodiments, thepresent invention relates to the treatment of anemia resulting from theuse of one or more renal replacement therapies, inclusive of dialysis,hemodialysis, peritoneal dialysis, hemofiltration, hemodiafiltration,and renal transplantation.

In some embodiments, the present invention relates to the treatment ofanemia in patients with chronic kidney disease who are not on dialysis.For instance, the present invention relates to patients in stage 1 CKD,or stage 2 CKD, or stage 3 CKD, or stage 4 CKD, or stage 5 CKD. In someembodiments, the present patient is stage 4 CKD or stage 5 CKD. In someembodiments, the present patient has undergone a kidney transplant. Insome embodiments, the present invention relates to the treatment ofanemia is a patient having an acute kidney injury (AKI).

In some embodiments, the anemia is induced by chemotherapy. Forinstance, the chemotherapy may be any myelosuppressive chemotherapy. Insome embodiment, the chemotherapy is one or more of Revlimid, Thalomid,dexamethasone, Adriamycin and Doxil. In some embodiments, thechemotherapy is one or more platinum-based drugs including cisplatin(e.g. PLATINOL) and carboplatin (e.g. PARAPLATIN). In some embodiments,the chemotherapy is any one of the chemotherapeutic agents describedherein. In some embodiments, the chemotherapy is any agent described inGroopman et al. J Natl Cancer Inst (1999) 91 (19): 1616-1634, thecontents of which are hereby incorporated by reference in theirentireties. In some embodiments, the present compositions and methodsare used in the treatment of chemotherapy-related anemia in later stagecancer patients (e.g. a stage IV, or stage III, or stage II cancer). Insome embodiments, the present compositions and methods are used in thetreatment of chemotherapy-related anemia in cancer patients receivingdose-dense chemotherapy or other aggressive chemotherapy regimens.

In some embodiments, the present invention relates to the treatment ofanemia in a patient having one or more blood-based cancers, such asleukemia, lymphoma, and multiple myeloma. Such cancers may affect thebone marrow directly. Further, the present invention relates tometastatic cancer that has spread to the bone or bone marrow. In someembodiments, the present invention relates to the treatment of anemia ina patient undergoing radiation therapy. Such radiation therapy maydamage the bone marrow, lowering its ability to make red blood cells. Infurther embodiments, the present invention relates to the treatment ofanemia in a patient having a reduction or deficiency of one or more ofiron, vitamin B12, and folic acid. In further embodiments, the presentinvention relates to the treatment of anemia in a patient havingexcessive bleeding including without limitation, after surgery or from atumor that is causing internal bleeding. In further embodiments, thepresent invention relates to the treatment of anemia in a patient havinganemia of chronic disease.

In some embodiments, the present methods and compositions stimulate redblood cell production. In some embodiments, the present methods andcompositions stimulate division and differentiation of committederythroid progenitors in the bone marrow.

Certain embodiments of the present invention are particularly useful fortreating chemotherapy-induced anemia in cancer patients. In someembodiments, the present methods and compositions allows for continuedadministration of the chimeric protein after a cancer patient'schemotherapy is finished. In some embodiments, the present methods andcompositions allows for treatment of a cancer patient without dosereduction relative to a non-cancer patient. In some embodiments, thepresent methods and compositions allows for treatment of a cancerpatient receiving chemotherapy and considered curable. In variousembodiments, the cancer patient has one or more of a history of bloodclots, recent surgery, prolonged periods of bed rest or limitedactivity, and treatment with a chemotherapeutic agent.

Kits

The invention also provides kits for the administration of any agentdescribed herein (e.g. the chimeric protein with or without variousadditional therapeutic agents). The kit is an assemblage of materials orcomponents, including at least one of the inventive pharmaceuticalcompositions described herein. Thus, in some embodiments, the kitcontains at least one of the pharmaceutical compositions describedherein.

The exact nature of the components configured in the kit depends on itsintended purpose. In one embodiment, the kit is configured for thepurpose of treating human subjects.

Instructions for use may be included in the kit. Instructions for usetypically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to treat cancer. Optionally, the kit also contains other usefulcomponents, such as, diluents, buffers, pharmaceutically acceptablecarriers, syringes, catheters, applicators, pipetting or measuringtools, bandaging materials or other useful paraphernalia as will bereadily recognized by those of skill in the art.

The materials and components assembled in the kit can be provided to thepractitioner stored in any convenience and suitable ways that preservetheir operability and utility. For example, the components can beprovided at room, refrigerated or frozen temperatures. The componentsare typically contained in suitable packaging materials. In variousembodiments, the packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging material may have an external label which indicates thecontents and/or purpose of the kit and/or its components.

Definitions

As used herein, “a,” “an,” or “the” can mean one or more than one.

Further, the term “about” when used in connection with a referencednumeric indication means the referenced numeric indication plus or minusup to 10% of that referenced numeric indication. For example, thelanguage “about 50” covers the range of 45 to 55.

An “effective amount,” when used in connection with medical uses is anamount that is effective for providing a measurable treatment,prevention, or reduction in the rate of pathogenesis of a disease ofinterest.

As used herein, something is “decreased” if a read-out of activityand/or effect is reduced by a significant amount, such as by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, or more, up to and including at least about 100%, inthe presence of an agent or stimulus relative to the absence of suchmodulation. As will be understood by one of ordinary skill in the art,in some embodiments, activity is decreased and some downstream read-outswill decrease but others can increase.

Conversely, activity is “increased” if a read-out of activity and/oreffect is increased by a significant amount, for example by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, or more, up to and including at least about 100% ormore, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, at least about10-fold, at least about 50-fold, at least about 100-fold, in thepresence of an agent or stimulus, relative to the absence of such agentor stimulus.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the compositions and methods of thistechnology. Similarly, the terms “can” and “may” and their variants areintended to be non-limiting, such that recitation that an embodiment canor may comprise certain elements or features does not exclude otherembodiments of the present technology that do not contain those elementsor features.

Although the open-ended term “comprising,” as a synonym of terms such asincluding, containing, or having, is used herein to describe and claimthe invention, the present invention, or embodiments thereof, mayalternatively be described using alternative terms such as “consistingof” or “consisting essentially of.”

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

The amount of compositions described herein needed for achieving atherapeutic effect may be determined empirically in accordance withconventional procedures for the particular purpose. Generally, foradministering therapeutic agents for therapeutic purposes, thetherapeutic agents are given at a pharmacologically effective dose. A“pharmacologically effective amount,” “pharmacologically effectivedose,” “therapeutically effective amount,” or “effective amount” refersto an amount sufficient to produce the desired physiological effect oramount capable of achieving the desired result, particularly fortreating the disorder or disease. An effective amount as used hereinwould include an amount sufficient to, for example, delay thedevelopment of a symptom of the disorder or disease, alter the course ofa symptom of the disorder or disease (e.g., slow the progression of asymptom of the disease), reduce or eliminate one or more symptoms ormanifestations of the disorder or disease, and reverse a symptom of adisorder or disease. Therapeutic benefit also includes halting orslowing the progression of the underlying disease or disorder,regardless of whether improvement is realized.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to about 50% ofthe population) and the ED50 (the dose therapeutically effective inabout 50% of the population). The dosage can vary depending upon thedosage form employed and the route of administration utilized. The doseratio between toxic and therapeutic effects is the therapeutic index andcan be expressed as the ratio LD50/ED50. In some embodiments,compositions and methods that exhibit large therapeutic indices arepreferred. A therapeutically effective dose can be estimated initiallyfrom in vitro assays, including, for example, cell culture assays. Also,a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 as determined in cellculture, or in an appropriate animal model. Levels of the describedcompositions in plasma can be measured, for example, by high performanceliquid chromatography. The effects of any particular dosage can bemonitored by a suitable bioassay. The dosage can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment.

In certain embodiments, the effect will result in a quantifiable changeof at least about 10%, at least about 20%, at least about 30%, at leastabout 50%, at least about 70%, or at least about 90%. In someembodiments, the effect will result in a quantifiable change of about10%, about 20%, about 30%, about 50%, about 70%, or even about 90% ormore. Therapeutic benefit also includes halting or slowing theprogression of the underlying disease or disorder, regardless of whetherimprovement is realized.

As used herein, “methods of treatment” are equally applicable to use ofa composition for treating the diseases or disorders described hereinand/or compositions for use and/or uses in the manufacture of amedicaments for treating the diseases or disorders described herein.This invention is further illustrated by the following non-limitingexamples.

EXAMPLES

Potential mutations in human interferon β that could lead to loss ofinteraction with IFNAR1 or IFNAR2, and for which the interaction andreceptor activation could be restored via an AcTakine (i.e. a chimera ofa signaling agent and a targeting agent) effect were chosen based onhomology modeling and structure analysis of related interferon receptorcomplexes.

Example 1 Identification of Interferon β Contact Residues forInteraction With IFNAR1

Homology models for the complex of human interferon β with theextracellular domain of IFNAR1 were constructed. For this model, thestructures with PDB codes 3WCY and 1AU1 were used as templates. 3 WCY isthe crystal structure of the complex of the extracellular domain ofmouse IFNAR1 with mouse interferon β. 1AU1 is the crystal structure ofhuman interferon β. The modeled sequences corresponded to UNIPROTentries P17181 for human IFNAR1 and P01574 for human interferon β. Thesequences were aligned with their templates using MAFFT (1), while humanand mouse interferon β were also aligned using structural superpositionin UCSF chimera (2).

The model for the human interferon β-IFNAR1 complex was built using amodeler (3), with the loopmodel class. 400 models were built, and themodel with the best DOPE score was chosen for further analysis. In thismodel, contacts between the human interferon β and IFNAR1 were visuallyinspected in UCSF chimera, and residues with side chain atoms within a 4Angström radius from the IFNAR1 chain were considered as possiblecontact residues. The accessible surface area of the complex model wasvisualized to detect those interferon β residues that were buried in themodel interface with IFNAR1.

In UCSF chimera, the model was structurally superposed with the crystalstructures with PDB id codes 3WCY, 3SE3 and 3SE4. 3SE3 is the crystalstructure of human IFNAR1 and IFNAR2 in complex met human interferon α2.3SE4 is the crystal structure of human IFNAR1 and IFNAR2 in complex withhuman interferon ω. The interferon—IFNAR1 interfaces and contacts in3WCY, 3SE3 and 3SE4 were also identified using visually inspection,accessible surface measurements and distance measurements in UCSFchimera, and via analysis with PDBsum (4), and PDBePISA (5).

Comparison of the interfaces and contacts in the model and in 3WCY, 3SE3and 3SE4 led to a list of contact residues and provided insight in theconservation of the contact in other interferons.

The lists of contacts were compared with literature data that studiedthe effects of mutations in interferon β (6) and interferon α2 (7-10) onIFNAR1 receptor binding, and on antiproliferative and antiviralactivities. Based on the comparison of the list with contact residues,the conservation of the contacts in other interferons and thisliterature data, we chose interferon β residues that most likelycontribute significantly to IFNAR1 binding. Based on this analysis, wedecided to mutate the side chain of these residues alone or incombination to alanine or glycine to remove the contacts, withoutintroducing steric hindrance.

Example 2 Identification of Interferon β Contact Residues forInteraction With IFNAR2

In UCSF chimera, the structure of human interferon β (pdb code 1AU1) wassuperposed on the structure of the complex of interferon α2 with IFNAR1and IFNAR2 (pdb code 3SE3). In this superposition, the residues ofinterferon α2 and interferon β that contact the IFNAR2 chain wereidentified. In UCSF chimera, the solvent accessible surface area of thecomplex in 3SE was calculated to visualize the interferon α2 residuesthat are buried in the interface with IFNAR2.

These contact residues in interferon α2 and possible contact residues ininterferon β for interaction with IFNAR2 were compared with reportedmutagenesis studies of interferon α2 (10-12) and interferon β (6).

By combining this information the interferon β residues that were mostimportant for binding to IFNAR2 were predicted. Based on this analysis,the side chain of these residues to alanine or glycine were mutated toremove the contacts, without introducing steric hindrance.

Example 3 Construction and Characterization of IFN-β-Containing Chimera

The following mutations are made, without wishing to be bound by theory,to affect interaction with IFNAR2: R152G; R152A; L151G+R152A; V148G;V148G+R152A; Y155G; R35G; R35A; L32G; L32A; L32A+R35A; W22G; andW22G+R27G.

The following mutations are made, without wishing to be bound by theory,to affect interaction with IFNAR1: L88G+Y92G; Y92G+I95A+N96G; K123GR124G; K123G; F67G; F67G+L88G+Y92G; F67S+L88S+Y92S; and F67G+R71A.

Combinations of mutations from the above are used to generate, withoutwishing to be bound by theory, mutations that affect interaction withIFNAR1 and IFNAR2.

Chimeric constructs are constructed as described in WO 2013/107791, theentire contents of which are hereby incorporated by reference, seeEXAMPLES Materials & Methods and Example 1).

Functional characterization is undertaken as in Example 2-10 WO2013/107791, the entire contents of which are hereby incorporated byreference.

Specifically, to generate chimeras (i.e., AcTaferons) based on humanIFNβ, the cytokine was fused via a 20*Gly-Gly-Ser flexible linker to aVHH targeting human CD20. The VHH employed in this example wasanti-human CD20 VHH (2HCD25), SEQ ID NO: 211. A total of 18 residues (ora combination thereof) believed to be involved in IFNAR1 or IFNAR2receptor binding were mutated (see list below). Effect of thesemutations and the CD20 targeting efficiency was evaluated by comparingthe anti-proliferative activity on Daudi (CD20 positive) and Wish (CD20negative) cells. Specifically, the following IFNβ-containing chimeraswere characterized:

Wild Type hIFNβ Coupled to Anti-huCD20 VHH (Without Wishing to be Boundby Theory):

-   -   1. P-126: hIFNβ

Mutants that Affect Interaction With IFNAR2 (Without Wishing to be Boundby Theory):

-   -   2. P-166: hIFNβ R152A    -   3. P-167: hIFNβ L151G-R152A    -   4. P-169: hIFNβ V148G-R152A    -   5. P-170: hIFNβ Y155G    -   6. P-171: hIFNβ R35G    -   7. P-172: hIFNβ R35A    -   8. P-173: hIFNβ L32G    -   9. P-174: hIFNß L32A    -   10. P-175: hIFNβL32A-R35A    -   11. P-176: hIFNβ W22G    -   12. P-177: hIFNβ W22G-R27G

Mutants that Affect Interaction With IFNAR1 (Without Wishing to be Boundby Theory):

-   -   13. P-178: hIFNβ L88G-Y92G    -   14. P-179: hIFNβ Y92G-I95A-N96G    -   15. P-180: hIFNβ K123G-R124G    -   16. P-181: hIFNβ K123G    -   17. P-182: hIFNβ F67G    -   18. P-183: hIFNβ F67G-L88G-Y92G    -   19. P-184: hIFNβ F67S-L88S-Y92S    -   20. P-185: hIFNβ F67G-R71A

The fusion protein huCD20 VHH-20*GGS-hulFNβ-6*His was cloned in thepCAGGS vector for mammalian expression. Mutants were made with standardsite-directed mutagenesis. Resulting constructs were transfected inHek293T cells using calcium phosphate and protein containingsupernatants was collected and used for analysis.

To measure the anti-proliferative effect of the resulting chimeras, Wish(2000 cells per 96-well) and Daudi (5000 cells per 96-well) cells wereseeded and cultured overnight. Cells were stimulated with a serialdilution of supernatants containing wildtype IFNβ, or chimeras for threedays. Proliferation was measured using the CellTiter-Glo luminescentcell viability assay according to the manufacturer's guidelines.

As shown in FIG. 1, the anti-proliferative activity of wild type IFNβwas comparable on Daudi and Wish cells. Linking the cytokine toanti-CD20 VHH clearly increased activity on Daudi cells (CD20 positive)compared to Wish cells (CD20 negative), thereby illustrating the CD20VHH targeting effect (24,29 fold). Some mutations hampered activity onWish cells but not Daudi cells. These mutations included: L151G/R152A;R35A; L32G; L32A; L32A/R35A; W22G; W22G/R27G; and F67G/R71A.Particularly, the L32A/R35A mutations resulted in a targeting effect ofup to 1050-fold, and the W22G/R275 mutations resulted in a targetingeffect of up to 1445-fold (see FIG. 1).

Example 4 Characterization of the Effects of IFN-β-Containing Chimerason STAT1 Phosphorylation

After the initial screening of the anti-proliferative effect of 18IFN-β-containing chimeras (i.e., “AcTaferons”) described in Example 3,seven mutants as listed below were further characterized:

Wild Type hIFN-β Coupled to Anti-huCD20 VHH:

-   -   1. P-126: hIFNβ

Mutants that Affect Interaction With IFNAR2 (Without Wishing to be Boundby Theory):

-   -   2. P-172: hIFNβ R35A    -   3. P-173: hIFNβ L32G    -   4. P-174: hIFNβ L32A    -   5. P-175: hIFNβ L32A-R35A    -   6. P-176: hIFNβ W22G    -   7. P-177: hIFNβ W22G-R27G

Mutants that Affect Interaction With IFNAR1 (Without Wishing to be Boundby Theory):

-   -   8. P-185: hIFNβ F67G-R17A

The effects of these mutations and the CD20 targeting efficiencies wereevaluated by comparing the ability to induce STAT1 phosphorylation in Bcells versus non-B cells in human peripheral blood mononuclear cells(PBMCs) by FACS. B cells were CD20 positive, but to avoid possibleinterference with the targeting anti-CD20 VHH, these cells wereidentified based on CD19 expression.

To generate the mutants, the fusion protein hCD20 VHH-20*GGS-hIFNβ-6*Hiswas cloned into the pCAGGS vector for mammalian expression. Mutants weremade with standard site-directed mutagenesis. Resulting constructs weretransfected in Hek293T cells using calcium phosphate. Proteins werepurified from the supernatants using the Talon metal affinity resin(Clontech) according to the manufacturer's guidelines and imidazole wasremoved from the samples using PD10 columns (GE Healthcare).

PBMCs from buffy coats of healthy donors were isolated using densitygradient centrifugation using Lymphoprep (StemCell technologies). Cellswere washed twice with FACS buffer (2% FBS, 1 mM EDTA in PBS) andstained with anti-human CD19 FITC (SinoBiologicals) for 20 minutes at 4°C. After two washes, cells were stimulated with a serial dilutionwildtype IFNβ or chimeras for 15 minutes at 37° C. After fixation (10minutes, 37° C., Fix Buffer I; BD Biosciences), permeabilisation (30minutes, on ice, Perm III Buffer I; BD Biosciences) and washing, cellswere stained with anti-STAT1 pY701 Ab (BD Biosciences). Samples wereacquired with a FACSCalibur (BD Biosciences) and analyzed with theFlowJo Version 10.2 software (LLC).

The ability of wildtype IFN-β to induce STAT1 phosphorylation wascomparable in CD19 positive and CD19 negative cells. Linking thecytokine to an anti-CD20 VHH clearly increased activity on CD19 positivecells compared to CD19 negative cells, thereby illustrating the CD20 VHHtargeting effect (7.1 fold). Most mutations clearly reduced activity onCD19 negative but not on CD19 positive cells. Particularly, theW22G-R27G mutations resulted in a targeting effect of up to 367-fold,and the L32A-R35A mutations resulted in a targeting effect of up to430-fold. The EC₅₀ values (ng/ml) for pSTAT1 in CD19 positive and CD19negative cells are summarized in the Table below (commas representdecimal points). The targeting effect was calculated by dividing theEC₅₀ values on CD19 negative cells by that on CD19 positive cells.

EC50 (ng/ml) Targeting CD19 neg CD19 post effect wt IFN beta 0.59220.4223 wt AFN beta 687.1 96.96 7.1 IFNAR2 AFN beta R35A (P-172) 908.21.339 678.3 binding AFN beta L32G (P-173) 2275 20.93 108.7 AFN beta L32A(P-174) 314.8 6.933 45.4 AFN beta 912.6 2.12 430.5 L32A-R35A (P-175) AFNbeta W22G (P-176) 203.1 0.9126 222.6 AFN beta 676.2 1.838 367.9W22G-R27G (P-177) IFNAR1 AFN beta F67G-R17A 327.9 25.49 12.9 binding

FIG. 2 shows the effects of various IFN-β-containing chimeras on STAT1phosphorylation. Mean fluorescence intensities of pSTAT1 in CD19positive and CD19 negative cells were plotted for wildtype IFN-β (i.e.SEQ ID NO: 1) or various IFNβ-containing chimeras (“AcTaferons,” PanelsA-I). For clarity, panel A is the mean fluorescence intensities ofpSTAT1 in CD19 positive and CD19 negative cells for wildtype IFN-β.Panel B is the mean fluorescence intensities of pSTAT1 in CD19 positiveand CD19 negative cells for wildtype IFN-β fused to anti-CD20 VHH.Panels C-I show the mean fluorescence intensities of pSTAT1 in CD19positive and CD19 negative cells for various mutant IFN-β fused toanti-CD20 VHH.

FIG. 2, panels J-K show the mean fluorescence intensities of pSTAT1 forwildtype IFNβ or IFNβ-containing chimeras in CD19 positive cellsrelative to CD19 negative cells.

FIG. 2, panels L-M show the targeting effects of various IFNβ-containingchimeras relative to wildtype IFN-β.

FIG. 2, panels N-O show the targeting effects of various mutantIFNβ-containing chimeras relative to wildtype IFNβ-containing chimeras(a fusion of a VHH against CD20 with wild type human IFN-β (SEQ ID NO:1)).

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

REFERENCES

1. Katoh K & Standley D M (2013) MAFFT multiple sequence alignmentsoftware version 7: improvements in performance and usability. Mol BiolEvol 30(4):772-780.

2. Pettersen E F, et al. (2004) UCSF Chimera-a visualization system forexploratory research and analysis. J Comput Chem 25(13):1605-1612.

3. Webb B & Sali A (2014) Comparative Protein Structure Modeling UsingMODELLER. Curr Protoc Bioinformatics 47:5 6 1-32.

4. de Beer T A, Berka K, Thornton J M, & Laskowski R A (2014) PDBsumadditions. Nucleic Acids Res 42(Database issue):D292-296.

5. Krissinel E & Henrick K (2007) Inference of macromolecular assembliesfrom crystalline state. J Mol Biol 372(3):774-797.

6. Runkel L, et al. (2000) Systematic mutational mapping of sites onhuman interferon-beta-1a that are important for receptor binding andfunctional activity. Biochemistry 39(10):2538-2551.

7. Hu R, Bekisz J, Schmeisser H, McPhie P, & Zoon K (2001) HumanIFN-alpha protein engineering: the amino acid residues at positions 86and 90 are important for antiproliferative activity. J Immunol167(3):1482-1489.

8. Pan M, et al. (2008) Mutation of the IFNAR-1 receptor binding site ofhuman IFN-alpha2 generates type I IFN competitive antagonists.Biochemistry 47(46):12018-12027.

9. Roisman L C, Jaitin D A, Baker D P, & Schreiber G (2005) Mutationalanalysis of the IFNAR1 binding site on IFNalpha2 reveals thearchitecture of a weak ligand-receptor binding-site. J Mol Biol353(2):271-281.

10. Piehler J, Roisman L C, & Schreiber G (2000) New structural andfunctional aspects of the type I interferon-receptor interactionrevealed by comprehensive mutational analysis of the binding interface.J Biol Chem 275(51):40425-40433.

11. Piehler J & Schreiber G (1999) Mutational and structural analysis ofthe binding interface between type I interferons and their receptorIfnar2. J Mol Biol 294(1):223-237.

12. Roisman L C, Piehler J, Trosset J Y, Scheraga H A, & Schreiber G(2001) Structure of the interferon-receptor complex determined bydistance constraints from double-mutant cycles and flexible docking.Proc Natl Acad Sci USA 98(23):13231-13236.

What is claimed is:
 1. A chimeric protein comprising: (a) a modifiedIFN-β, said modified IFN-β having one or more mutations that conferreduced affinity and/or reduced activity for or at interferon-α/βreceptor (IFNAR) as compared to a wild type IFN-β, and (b) one or moretargeting moieties, said targeting moieties comprising recognitiondomains which specifically bind to antigens or receptors of interest,wherein: the modified IFN-β and the one or more targeting moieties areoptionally connected with one or more linkers, the modified IFN-βcomprises: (i) Y92G, I95A, and N96G mutations, (ii) K123G and R124Gmutations, (iii) F67G, L88G, and Y92G mutations, or (iv) F67S, L88S, andY92S mutations, and the recognition domain is a single-domain antibodyor a single-chain antibody (scFv).
 2. The chimeric protein of claim 1,wherein the modified IFN-β exhibits reduced affinity for IFNAR1.
 3. Thechimeric protein of claim 1, wherein the modified IFN-β exhibits reducedaffinity for IFNAR2.
 4. The chimeric protein of claim 1, wherein the oneor more mutations confer reduced affinity that is restorable byattachment to one or more targeting moieties.
 5. The chimeric protein ofclaim 1, wherein the targeting moiety is directed against a tumor cellor an immune cell.
 6. The chimeric protein of claim 1, wherein therecognition domain is a V_(HH), humanized V_(HH), or camelized V_(HH).7. A recombinant nucleic acid composition encoding the chimeric proteinof claim
 1. 8. A host cell comprising a nucleic acid of claim
 7. 9. Achimeric protein comprising: (a) a modified IFN-β, said modified IFN-βhaving one or more mutations that confer reduced affinity and/or reducedactivity for or at interferon-α/β receptor (IFNAR) as compared to a wildtype IFN-β, and (b) one or more targeting moieties, said targetingmoieties comprising recognition domains which specifically bind toantigens or receptors of interest, wherein: the modified IFN-β and theone or more targeting moieties are optionally connected with one or morelinkers, the modified IFN-β comprises: (i) W22G and R27G mutations, (ii)L32A and R35A mutations, (iii) L151G and R152A mutations, or (iv) V148Gand R152A mutations, and the recognition domain is a single: domainantibody or a single-chain antibody (scFv).
 10. The chimeric protein ofclaim 9, wherein the modified IFN-β exhibits reduced affinity forIFNAR1.
 11. The chimeric protein of claim 9, wherein the modified IFN-βexhibits reduced affinity for IFNAR2.
 12. The chimeric protein of claim9, wherein the one or more mutations confer reduced affinity that isrestorable by attachment to one or more targeting moieties.
 13. Thechimeric protein of claim 9, wherein the targeting moiety is directedagainst a tumor cell or an immune cell.
 14. The chimeric protein ofclaim 9, wherein the recognition domain is a V_(HH), humanized V_(HH),or camelized V_(HH).
 15. A recombinant nucleic acid composition encodingthe chimeric protein of claim
 9. 16. A host cell comprising a nucleicacid of claim 15.